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Multiple sequence alignment
Tuesday, Feb 8 2011
Suggested installation for the following tools on your own computer:
ClustalX, Mega4, GeneDoc; treeview
Multiple sequence alignment
• to define what a multiple sequence alignment is and how it is generated; to describe profile HMMs
• to introduce databases of multiple sequence alignments
• to introduce ways you can make your own multiple sequence alignments
• to show how a multiple sequence alignment provides the basis for phylogenetic trees
Page 319
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
Multiple sequence alignment: definition
• a collection of three or more protein (or nucleic acid) sequences that are partially or completely aligned
• homologous residues are aligned in columns across the length of the sequences
• residues are homologous in an evolutionary sense
• residues are homologous in a structural sense
Page 320
ClustalW
Note how the region of a conserved histidine (▼) varies depending on which algorithm is used
Praline
MUSCLE
Probcons
TCoffee
Multiple sequence alignment: properties
• not necessarily one “correct” alignment of a protein family
• protein sequences evolve...
• ...the corresponding three-dimensional structures of proteins also evolve
• may be impossible to identify amino acid residues that align properly (structurally) throughout a multiple sequence alignment
• for two proteins sharing 30% amino acid identity, about 50% of the individual amino acids are superposable in the two structures
Page 320
Multiple sequence alignment: features
• some aligned residues, such as cysteines that form disulfide bridges, may be highly conserved
• there may be conserved motifs such as a transmembrane domain
• there may be conserved secondary structure features
• there may be regions with consistent patterns of insertions or deletions (indels)
Page 320
Multiple sequence alignment: uses
• MSA is more sensitive than pairwise alignment to detect homologs
• BLAST output can take the form of a MSA, and can reveal conserved residues or motifs
• Population data can be analyzed in a MSA (PopSet)
• A single query can be searched against a database of MSAs (e.g. PFAM)
• Regulatory regions of genes may have consensus sequences identifiable by MSA
Page 321
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
[6] Introduction to molecular evolution and phylogeny
Multiple sequence alignment: methods
Exact methods: dynamic programmingInstead of the 2-D dynamic programming matrix in theNeedleman-Wunsch technique, think about a 3-D,4-D or higher order matrix.
Exact methods give optimal alignments but are not feasible in time or space for more than ~10 sequences.
Still an extremely active field.
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
[6] Introduction to molecular evolution and phylogeny
Multiple sequence alignment: methods
Progressive methods: use a guide tree (a little like aphylogenetic tree but NOT a phylogenetic tree) to determine how to combine pairwise alignments one by oneto create a multiple alignment.
Making multiple alignments using trees was a verypopular subject in the ‘80s. Fitch and Yasunobu (1974)may have first proposed the idea, but Hogeweg andHesper (1984) and many others worked on the topic beforeFeng and Doolittle (1987)—they made one important contribution that got their names attached to thisalignment method.
Examples: CLUSTALW, MUSCLE
Multiple sequence alignment: methods
Example of MSA using ClustalW: two data sets
Five distantly related lipocalins (human to E. coli)
Five closely related RBPs
When you do this, obtain the sequences of interest in the FASTA format! (You can save them in a Word document)
Page 321
The input for ClustalW: a group of sequences(DNA or protein) in the FASTA format
Get sequences from Entrez Protein (or HomoloGene)
You can display sequences from Entrez Protein in the fasta format
When you get a DNA sequence from Entrez Nucleotide, you can click CDS to select only the
coding sequence.
This is very useful for phylogeny studies.
HomoloGene: an NCBI resource to obtain multiple related sequences
[1] Enter a query at NCBI such as globin[2] Click on HomoloGene (left side)[3] Choose a HomoloGene family, and view in the fasta format
Use ClustalW to do a progressive MSA
http://www2.ebi.ac.uk/clustalw/ Fig. 10.1
Page 321
Feng-Doolittle MSA occurs in 3 stages
[1] Do a set of global pairwise alignments (Needleman and Wunsch’s dynamic programming
algorithm)
[2] Create a guide tree
[3] Progressively align the sequences
Page 321
Progressive MSA stage 1 of 3:generate global pairwise alignments
Fig. 10.2Page 323
five distantly related lipocalins
best score
Progressive MSA stage 1 of 3:generate global pairwise alignments
Start of Pairwise alignmentsAligning...Sequences (1:2) Aligned. Score: 84Sequences (1:3) Aligned. Score: 84Sequences (1:4) Aligned. Score: 91Sequences (1:5) Aligned. Score: 92Sequences (2:3) Aligned. Score: 99Sequences (2:4) Aligned. Score: 86Sequences (2:5) Aligned. Score: 85Sequences (3:4) Aligned. Score: 85Sequences (3:5) Aligned. Score: 84Sequences (4:5) Aligned. Score: 96
Fig. 10.4Page 325
five closely related lipocalins
best score
Number of pairwise alignments needed
For n sequences, (n-1)(n) / 2
For 5 sequences, (4)(5) / 2 = 10
Page 322
Feng-Doolittle stage 2: guide tree
• Convert similarity scores to distance scores
• A tree shows the distance between objects
• Use UPGMA (defined in the phylogeny lecture)(Unweighted Pair Group Method with Arithmetic Mean)
• ClustalW provides a syntax to describe the tree
• A guide tree is not a phylogenetic tree
Page 323
Progressive MSA stage 2 of 3:generate a guide tree calculated from
the distance matrix
Fig. 10.2Page 323
Progressive MSA stage 2 of 3:generate guide tree
((gi|5803139|ref|NP_006735.1|:0.04284,(gi|6174963|sp|Q00724|RETB_MOUS:0.00075,gi|132407|sp|P04916|RETB_RAT:0.00423):0.10542):0.01900,gi|89271|pir||A39486:0.01924,gi|132403|sp|P18902|RETB_BOVIN:0.01902);
Fig. 10.4Page 325
five closely related lipocalins
Feng-Doolittle stage 3: progressive alignment
• Make a MSA based on the order in the guide tree
• Start with the two most closely related sequences
• Then add the next closest sequence
• Continue until all sequences are added to the MSA
• Rule: “once a gap, always a gap.”
Page 324
Progressive MSA stage 3 of 3:progressively align the sequences
following the branch order of the tree
Fig. 10.3Page 324
Progressive MSA stage 3 of 3:CLUSTALX output
Note that you can download CLUSTALX locally, rather than using a web-based program!
Clustal W alignment of 5 closely related lipocalins
CLUSTAL W (1.82) multiple sequence alignment
gi|89271|pir||A39486 MEWVWALVLLAALGSAQAERDCRVSSFRVKENFDKARFSGTWYAMAKKDP 50gi|132403|sp|P18902|RETB_BOVIN ------------------ERDCRVSSFRVKENFDKARFAGTWYAMAKKDP 32gi|5803139|ref|NP_006735.1| MKWVWALLLLAAW--AAAERDCRVSSFRVKENFDKARFSGTWYAMAKKDP 48gi|6174963|sp|Q00724|RETB_MOUS MEWVWALVLLAALGGGSAERDCRVSSFRVKENFDKARFSGLWYAIAKKDP 50gi|132407|sp|P04916|RETB_RAT MEWVWALVLLAALGGGSAERDCRVSSFRVKENFDKARFSGLWYAIAKKDP 50 ********************:* ***:*****
gi|89271|pir||A39486 EGLFLQDNIVAEFSVDENGHMSATAKGRVRLLNNWDVCADMVGTFTDTED 100gi|132403|sp|P18902|RETB_BOVIN EGLFLQDNIVAEFSVDENGHMSATAKGRVRLLNNWDVCADMVGTFTDTED 82gi|5803139|ref|NP_006735.1| EGLFLQDNIVAEFSVDETGQMSATAKGRVRLLNNWDVCADMVGTFTDTED 98gi|6174963|sp|Q00724|RETB_MOUS EGLFLQDNIIAEFSVDEKGHMSATAKGRVRLLSNWEVCADMVGTFTDTED 100gi|132407|sp|P04916|RETB_RAT EGLFLQDNIIAEFSVDEKGHMSATAKGRVRLLSNWEVCADMVGTFTDTED 100 *********:*******.*:************.**:**************
gi|89271|pir||A39486 PAKFKMKYWGVASFLQKGNDDHWIIDTDYDTYAAQYSCRLQNLDGTCADS 150gi|132403|sp|P18902|RETB_BOVIN PAKFKMKYWGVASFLQKGNDDHWIIDTDYETFAVQYSCRLLNLDGTCADS 132gi|5803139|ref|NP_006735.1| PAKFKMKYWGVASFLQKGNDDHWIVDTDYDTYAVQYSCRLLNLDGTCADS 148gi|6174963|sp|Q00724|RETB_MOUS PAKFKMKYWGVASFLQRGNDDHWIIDTDYDTFALQYSCRLQNLDGTCADS 150gi|132407|sp|P04916|RETB_RAT PAKFKMKYWGVASFLQRGNDDHWIIDTDYDTFALQYSCRLQNLDGTCADS 150 ****************:*******:****:*:* ****** *********
Fig. 10.5Page 326
* asterisks indicate identity in a column
Progressive MSA stage 3 of 3:progressively align the sequences
following the branch order of the tree:Order matters
THE LAST FAT CAT THE FAST CAT THE VERY FAST CAT THE FAT CAT
THE LAST FAT CATTHE FAST CAT ---
THE LAST FA-T CATTHE FAST CA-T ---THE VERY FAST CAT THE LAST FA-T CAT
THE FAST CA-T ---THE VERY FAST CATTHE ---- FA-T CATAdapted from C. Notredame, Pharmacogenomics 2002
Progressive MSA stage 3 of 3:progressively align the sequences
following the branch order of the tree:Order matters
THE FAT CAT THE FAST CAT THE VERY FAST CAT THE LAST FAT CAT
THE FA-T CATTHE FAST CAT
THE ---- FA-T CATTHE ---- FAST CATTHE VERY FAST CAT THE ---- FA-T CAT
THE ---- FAST CATTHE VERY FAST CATTHE LAST FA-T CATAdapted from C. Notredame, Pharmacogenomics 2002
Why “once a gap, always a gap”?
• There are many possible ways to make a MSA
• Where gaps are added is a critical question
• Gaps are often added to the first two (closest) sequences
• To change the initial gap choices later on would beto give more weight to distantly related sequences
• To maintain the initial gap choices is to trustthat those gaps are most believable
Page 324
Additional features of ClustalW improveits ability to generate accurate MSAs
• Individual weights are assigned to sequences; very closely related sequences are given less weight,while distantly related sequences are given more weight
• Scoring matrices are varied dependent on the presenceof conserved or divergent sequences, e.g.:
PAM20 80-100% idPAM60 60-80% idPAM120 40-60% idPAM350 0-40% id
• Residue-specific gap penalties are applied
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
[6] Introduction to molecular evolution and phylogeny
Multiple sequence alignment: methods
Iterative methods: compute a sub-optimal solution and keep modifying that intelligently using dynamic programming or other methods until the solution converges.
Examples: IterAlign, Praline, MAFFT
MUSCLE: next-generation progressive MSA
[1] Build a draft progressive alignment
Determine pairwise similarity through k-mer counting (not by alignment)
Compute distance (triangular distance) matrix
Construct tree using UPGMA
Construct draft progressive alignment following tree
MUSCLE: next-generation progressive MSA
[2] Improve the progressive alignment
Compute pairwise identity through current MSA
Construct new tree with Kimura distance measures
Compare new and old trees: if improved, repeat this step, if not improved, then we’re done
MUSCLE: next-generation progressive MSA
[3] Refinement of the MSA
Split tree in half by deleting one edge
Make profiles of each half of the tree
Re-align the profiles
Accept/reject the new alignment
http://www.ebi.ac.uk/muscle/
MUSCLE output (formatted with SeaView)
SeaView is a graphical multiple sequence alignment editor available at http://pbil.univ-lyon1.fr/software/seaview.html
Praline output for the same alignment: pure iterative approach
Boxes highlight a region that is difficult to align
Iterative approaches: MAFFT
• Uses Fast Fourier Transform to speed up profile alignment
• Uses fast two-stage method for building alignments using k-mer frequencies
• Offers many different scoring and aligning techniques• One of the more accurate programs available• Available as standalone or web interface• Many output formats, including interactive
phylogenetic trees
Iterative approaches: MAFFT
Has about 1000 advanced settings!
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
[6] Introduction to molecular evolution and phylogeny
Multiple sequence alignment: methods
Consistency-based algorithms: generally use a database of both local high-scoring alignments and long-range global alignments to create a final alignment
These are very powerful, very fast, and very accurate methods
Examples: T-COFFEE, Prrp, DiAlign, ProbCons
ProbCons—consistency-based approach
Combines iterative and progressive approaches with a unique probabilistic model.
Uses Hidden Markov Models (more in a minute) to calculate probability matrices for matching residues, uses this to construct a guide tree
Progressive alignment hierarchically along guide tree
Post-processing and iterative refinement (a little like MUSCLE)
2e Fig. 5.12
ProbCons—consistency-based approach
Sequence x xi
Sequence y yj
Sequence z zk
If xi aligns with zk
and zk aligns with yj
then xi should align with yj
ProbCons incorporates evidence from multiple sequences to guide the creation of a pairwise alignment.
ProbCons—consistency-based approachhttp://probcons.stanford.edu/
ProbCons output for the same alignment: consistency iteration helps
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
[6] Introduction to molecular evolution and phylogeny
http://tcoffee.org
Make an MSAMSA w. structural dataCompare MSA methodsMake an RNA MSACombine MSA methods
Consistency-basedStructure-based
Back translate protein MSA
APDB ClustalW output
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
[6] Introduction to molecular evolution and phylogeny
Multiple sequence alignment: methods
How do we know which program to use?
There are benchmarking multiple alignment datasets that have been aligned painstakingly by hand, by structural similarity, or by extremely time- and memory-intensive automated exact algorithms.
Some programs have interfaces that are more user-friendly than others. And most programs are excellent so it depends on your preference.
If your proteins have 3D structures, use these to help you judge your alignments. For example, try Expresso at http://www.tcoffee.org.
[1] Create or obtain a database of protein sequencesfor which the 3D structure is known. Thus we candefine “true” homologs using structural criteria.
[2] Try making multiple sequence alignmentswith many different sets of proteins (very related,very distant, few gaps, many gaps, insertions,outliers).
[3] Compare the answers.
Strategy for assessment of alternativemultiple sequence alignment algorithms
Page 346
Multiple sequence alignment: methods
Benchmarking tests suggest that ProbCons, a consistency-based/progressive algorithm, performs the best on the BAliBASE set, although MUSCLE, a progressive alignment package, is an extremely fast and accurate program.
ClustalW is the most popular program. It has a nice interface (especially with ClustalX) and is easy to use.
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
[6] Introduction to molecular evolution and phylogeny
Multiple sequence alignment to profile HMMs
► Hidden Markov models (HMMs) are “states”that describe the probability of having aparticular amino acid residue at arrangedin a column of a multiple sequence alignment
► HMMs are probabilistic models
► HMMs may give more sensitive alignmentsthan traditional techniques such as progressive alignment
Page 325
Simple Hidden Markov Model
Observation: YNNNYYNNNYN
(Y=goes out, N=doesn’t go out)
What is underlying reality (the hidden state chain)?
R
S
0.15
0.85
0.2
0.8
P(dog goes out in rain) = 0.15
P(dog goes out in sun) = 0.85
GTWYA (hs RBP)GLWYA (mus RBP)GRWYE (apoD)GTWYE (E Coli)GEWFS (MUP4)
An HMM is constructed from a MSA
Example: five lipocalins
Fig. 10.6Page 327
GTWYAGLWYAGRWYEGTWYEGEWFS
PositionProb. 1 2 3 4 5p(G) 1.0p(T) 0.4p(L) 0.2p(R) 0.2p(E) 0.2 0.4p(W) 1.0p(Y) 0.8p(F) 0.2p(A) 0.4p(S) 0.2
Fig. 10.6Page 327
GTWYAGLWYAGRWYEGTWYEGEWFS
Prob. 1 2 3 4 5p(G) 1.0p(T) 0.4p(L) 0.2p(R) 0.2p(E) 0.2 0.4p(W) 1.0p(Y) 0.8p(F) 0.2p(A) 0.4p(S) 0.2
P(GEWYE) = (1.0)(0.2)(1.0)(0.8)(0.4) = 0.064
log odds score = ln(1.0) + ln(0.2) + ln(1.0) + ln(0.8) + ln(0.4) = -2.75 Fig. 10.6Page 327
GTWYAGLWYAGRWYEGTWYEGEWFS
P(GEWYE) = (1.0)(0.2)(1.0)(0.8)(0.5) = 0.08
log odds score = ln(1.0) + ln(0.2) + ln(1.0) + ln(0.8) + ln(0.5) = -2.53
G:1.0 W:1.0
T:0.4
L:0.2
R:0.2
E:0.2
Y:0.8
F:0.2
A:0.5
E:0.5
S:1.0
Structure of a hidden Markov model (HMM)
M
Iy
Ix
p1
p7
p6
p5
p3
p2
p4
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
[6] Introduction to molecular evolution and phylogeny
Two kinds of multiple sequence alignment resources
Text-based or query-based searches:CDD, Pfam (profile HMMs), PROSITE
[2] Multiple sequence alignment by manual input
Muscle, ClustalW, ClustalX
[1] Databases of multiple sequence alignments
Page 329
BLOCKSCDD Pfam SMARTDOMO (Gapped MSA)INTERPROiProClassMetaFAMPRINTSPRODOM (PSI-BLAST)PROSITE
Databases of multiple sequence alignments
TheseUseHMMs
PFAM (protein family) database:http://pfam.sanger.ac.uk/
Fig. 10.11Page 331
PFAM (protein family) text search result
Fig. 10.12Page 334
PFAM HMM for lipocalins
20 amino acids
position
PFAM HMM for lipocalins: GXW motif
G W
20 amino acids
PFAM GCG MSF format
Fig. 10.13Page 335
Pfam (protein family) database
PFAM JalView viewer
Fig. 10.14Page 336
PFAM JalView viewer
Fig. 10.15Page 336
PFAM JalView viewer:principalcomponentsanalysis(PCA)
Fig. 10.16Page 337
Fig. 10.17Page 337
SMART: Simple ModularArchitecture Research Tool(emphasis on cell signaling)
Page 338
SMART: lipocalin result
Fig. 10.18Page 338
BLOCKSCDD Pfam SMARTDOMO (Gapped MSA)INTERPROiProClassMetaFAMPRINTSPRODOM (PSI-BLAST)PROSITE
Databases of multiple sequence alignments
ConservedDomainDatabase(CDD) at NCBI = PFAM + SMART
[1] Go to NCBI Structure[2] Click CDD[3] Enter a text query, or a protein sequence
CDD: Conserved domain database
CDD: Conserved domain database
CDD=
PFAM+
SMART
Fig. 10.20Page 339
Purpose: to find conserved domainsin the query sequence
Query = your favorite protein
Database = set of many position-specificscoring matrices (PSSMs), i.e. a set of MSAs
CDD is related to PSI-BLAST, but distinct
CDD searches against profiles generatedfrom pre-selected alignments
CDD uses RPS-BLAST: reverse position-specific
Page 333
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
[6] Introduction to molecular evolution and phylogeny
MEGA version 4: Molecular Evolutionary Genetics Analysis
Download from www.megasoftware.net
MEGA version 4: Molecular Evolutionary Genetics Analysis
MEGA version 4: Molecular Evolutionary Genetics Analysis
Two ways to create a multiple sequence alignment1. Open the Alignment Explorer, paste in a FASTA MSA2. Select a DNA query, do a BLAST search
Once your sequences are in MEGA, you can run ClustalWthen make trees and do phylogenetic analyses
1
2
[1] Open the Alignment Explorer
[2] Select “Create a new alignment”
[3] Click yes (for DNA) or no (for protein)
[4] Find, select, and copy a multiple sequence alignment (e.g. from Pfam; choose FASTA with dashes for gaps)
[5] Paste it into MEGA
[6] If needed, run ClustalW to align the sequences
[7] Save (Ctrl+S) as .masthen exit and save as .meg
Multiple sequence alignment: outline
[1] Introduction to MSAExact methodsProgressive (ClustalW)Iterative (MUSCLE)Consistency (ProbCons)Structure-based (Expresso)Conclusions: benchmarking studies
[3] Hidden Markov models (HMMs), Pfam and CDD
[4] MEGA to make a multiple sequence alignment
[5] Multiple alignment of genomic DNA
[6] Introduction to molecular evolution and phylogeny
Multiple sequence alignment of genomic DNA
There are typically few sequences (up to several dozen, each having up to millions of base pairs. Adding more species improves accuracy.
Alignment of divergent sequences often reveals islands of conservation (providing “anchors” for alignment).
Chromosomes are subject to inversions, duplications, deletions, and translocations (often involving millions of base pairs). E.g. human chromosome 2 is derived from the fusion of two acrocentric chromosomes.
There are no benchmark datasets available.