1 Introduction to Bioinformatics Online Course: IBT Multiple Sequence Alignment Building Multiple Sequence Alignment Lec1 Building a Multiple Sequence Alignment
2 Learning Outcomes 1- Understanding Why multiple sequence alignment is useful for scientists 2-Identifying situations where multiple alignments do not help 3-Main Criteria for Building a Multiple Sequence Alignment 4- Main Applications of Multiple Sequence Alignments 5-What are the kinds of sequences you re looking for? 6- Tips for Naming sequences 7- Tips for difficult MSA to interpret 8- Comparing sequences you cannot align
3 In the coming lectures we will learn 1- Gathering the sequences you need to make a multiple sequence alignment 2- Differences between some famous multiple sequence alignment programs COBALT (Constraint-based Multiple Alignment Tool) New ClustalW (everybody uses it), MUSCLE (very fast) TCOFFEE (accurate and combine sequences and structures) 3- Creating and comparing multiple sequence alignments with - Comparing sequences you cannot align Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
4 Building a Multiple Sequence Alignment (1)
5 - In many ways, multiple sequence alignments are to bioinformatics what Swiss knives are to MacGyver - Building multiple sequence alignments is far from an exact science - In fact, it s more art than science, requiring that you use everything you know in bioinformatics and in biology. Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
6 Identifying situations where multiple alignments do not help
7 Don t work well for assembling the sequence pieces in a sequencing project. if you want to turn an EST cluster into a gene sequence When the sequence you re interested in has no homologue in any of the sequence databases (in this case you can use functional criteria and conducting a pattern search). Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
8 Building informative alignments Gathering your sequences Compute a multiple sequence alignment Evaluate the quality of your alignment Interpreting Your MSA keep the sequences for further analysis Mansour A, Jaime A. Teixeira da Silva, Gábor Gyulai ) 2009( Assessment of molecular (dis)similarity: The role of multiple sequence alignments (MSA) programs in biological research. Genes, genomes and genomics ( :)1 eussi laiceps(3. Print ISSN ( ) Bioinformatics SI.)
9 What we are looking for with MSA? The idea behind a multiple alignment is to put amino acids or nucleotides in the same column because they re similar according to some criterion. You can use four major criteria to build a multiple alignment of sequences that all have different properties. Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
10 Main Criteria for Building a Multiple Sequence Alignment 1- Structural similarity Amino acids that play the same role in each structure are in the same column. Structure-superposition programs are the only ones that use this criterion.
11 Main Criteria for Building a Multiple Sequence Alignment 2- Evolutionary similarity Amino acids or nucleotides related to the same amino acid (or nucleotide) in the common ancestor of all the sequences are put in the same column. No automatic program explicitly uses this criterion, but they all try to deliver an alignment that respects it. Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
12 Main Criteria for Building a Multiple Sequence Alignment 3- Functional similarity Amino acids or nucleotides with the same function are in the same column. No automatic program explicitly uses this criterion, but if the information is available, you can force some programs to respect it or you can edit your alignment manually. Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
13 Main Criteria for Building a Multiple Sequence Alignment 4- Sequence similarity Amino acids in the same column are those that yield an alignment with maximum similarity. Most programs use sequence similarity because it is the easiest criterion. When the sequences are closely related, their structural, evolutionary, and functional similarities are equivalent to sequence similarity. Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
14 Main Applications of Multiple Sequence Alignments
15 Main Applications of Multiple Sequence Alignments Extrapolation A good multiple alignment can help convince you that an uncharacterized sequence is really a member of a protein family. Alignments that include Swiss-Prot sequences are the most informative. Use the ExPASyBLAST server (at www. expasy.ch/tools/blast/) to gather and align them. Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
16 Main Applications of Multiple Sequence Alignments Phylogenetic Analysis If you carefully choose the sequences you include in your analysis multiple alignment, you can reconstruct the history of these proteins. Use the Pasteur Phylip server at bioweb.pasteur.fr/seqanal/phylogeny/phylip-uk.html. Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
17 Main Applications of Multiple Sequence Alignments Pattern identification By discovering very conserved positions, you can identify a identification region that is characteristic of a function (in proteins or in nucleic-acid sequences). Use the Weblogo server Paste your sequences here Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
18 Main Applications of Multiple Sequence Alignments Domain identification It is possible to turn a multiple sequence alignment into a profile that describes a protein family or a protein domain (PSSM). You can use this profile to scan databases for new members of the family. Use PROSITE ( Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
19 Main Applications of Multiple Sequence Alignments DNA regulatory elements You can turn a DNA multiple alignment of a binding site into a weight matrix and scan other DNA sequences for potentially elements similar binding sites. Use the Gibbs sampler to identify these sites: Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
20 Main Applications of Multiple Sequence Alignments Structure prediction A good multiple alignment can give you an almost perfect prediction of your protein secondary structure for both proteins and RNA. Sometimes it can also help in the building of a 3-D model. Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
21 Main Applications of Multiple Sequence Alignments nssnp analysis Various gene alleles often have different amino-acid sequences. Multiple alignments can help you predict whether a Non- Synonymous Single-Nucleotide Polymorphism is likely to be harmful. See the SIFT site for more details: Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
22 Main Applications of Multiple Sequence Alignments PCR analysis A good multiple alignment can help you identify the less degenerated portions of a protein family, in order to fish out new members by PCR (polymerase chain reaction). If this is what you want to do, you can use the following site: blocks.fhcrc.org/codehop.html Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
23 Paste your sequences here
24 What are the kinds of sequences you re looking for? Always bear in mind that in evolution: 1- Important amino acids (or nucleotides) are NOT allowed to mutate. For instance, active sites of enzymes are much conserved. 2- Less-important residues change more easily sometimes randomly and sometimes in order to adapt a function. Claverie J, Notredame C (2007). Bioinformatics for Dummies (2 nd Edn). Wiley publishing, Inc. 436 pp.
25 Tips for Naming sequences Never use white spaces Do not use special symbols Never use names longer than 15 characters Never give the same name to two different sequences Mansour A, Jaime A. Teixeira da Silva, Gábor Gyulai ) 2009( Assessment of molecular (dis)similarity: The role of multiple sequence alignments (MSA) programs in biological research. Genes, genomes and genomics ( :)1 eussi laiceps(3. Print ISSN ( ) Bioinformatics SI.)
26 Tips for difficult MSA to interpret 1 Remove Click insertions/deletions to add Title 2 13 Click to add Title Redo MSA with the smaller set Click to add Title Keep trimming to interpret Mansour A, Jaime A. Teixeira da Silva, Gábor Gyulai ) 2009( Assessment of molecular (dis)similarity: The role of multiple sequence alignments (MSA) programs in biological research. Genes, genomes and genomics ( :)1 eussi laiceps(3. Print ISSN ( ) Bioinformatics SI.)
27 Enhancing Alignments Remove gaps Remove extremities Keep informative blocks Enhancing your Alignment Mansour A, Jaime A. Teixeira da Silva, Gábor Gyulai ) 2009( Assessment of molecular (dis)similarity: The role of multiple sequence alignments (MSA) programs in biological research. Genes, genomes and genomics ( :)1 eussi laiceps(3. Print ISSN ( ) Bioinformatics SI.)
28 (to try in your own time) Searching sequences on the ExPASy server Only to retrieve protein sequences in FASTA format Example: Heat shock factor 1 (HSF1) Choose
30 (to try in your own time) Select the sequences you want This is the most delicate part of the process you can use the following guidelines - Select the top sequence. - For a first analysis, you want to select ten sequences or fewer. check it s similar to the query sequence - along its entire length.
31 (to try in your own time) Methods to export your sequences FASTA: Generates a file that contains your sequences in FASTA format. ClustalW, Tcoffee, and MAFFT: These are MSA packages running on the EMBnet server. Reduce Redundancy: This option will extract the most meaningful sequences from your dataset. Pratt: Will search for conserved motifs in your sequences without aligning them
32 Practical (to try in your own time) - Go to - Search for HSF1 - Click on (UniProtKB) - Retrieve your protein sequences (eg. Heat shock Factor1 HSF1 ) from different organisms - This will take you to - Select your organism (Human, Rat, Mouse, Arabidopsis, Chicken, Pig) - Click Download (Download Selected) then (Go) - Save it in FASTA format in one text file. - Align the sequences using Clustal Omega - Checking the gene-based phylogentics tree - Add one more sequence NOT related sequence (Out Group) - Checking the change on the gene-based phylogentics tree
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