1. Multiple sequence alignment
Tutorial 5
Multiple sequence alignment

2. Multiple Sequence Alignment – When?
More than two sequences
DNA
Protein
Evolutionary relation
Homology  Phylogenetic tree
Detect motif
GTCGTAGTCGGCTCGAC GTCTAGCGAGCGTGAT GCGAAGAGGCGAGC GCCGTCGCGTCGTAAC
GTCGTAGTCG-GC-TCGAC GTC-TAG-CGAGCGT-GAT GC-GAAG-AG-GCG-AG-C GCCGTCG-CG-TCGTA-AC A D B C

3. Multiple Sequence Alignment – How?
Dynamic Programming
Optimal alignment
Exponential in #Sequences
Progressive
Efficient
Heuristic
GTCGTAGTCGGCTCGAC GTCTAGCGAGCGTGAT GCGAAGAGGCGAGC GCCGTCGCGTCGTAAC
GTCGTAGTCG-GC-TCGAC GTC-TAG-CGAGCGT-GAT GC-GAAG-AG-GCG-AG-C GCCGTCG-CG-TCGTA-AC A D B C

4. Hierarchical Clustering
A way to represent similarities graphically.
Sums up a pairwise distance matrix as a dendrogram.
Not all matrices can be embedded in a tree without error.
TGTTAAC
TGT-AAC
TGT--AC
ATGT--C
ATGTGGC

5. ClustalW Pairwise alignment – calculate distance matrix Guided tree
Progressive alignment using the guide tree
“CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice”, J D Thompson et al

6. Progressive (incremental)
ClustalW
Progressive (incremental)
At each step align two existing alignments or sequences.
Gaps present in older alignments remain fixed.
Uses the Neighbor Joining algorithm.

7. Neighbor Joining Algorithm
An agglomerative hierarchical clustering method.
Constructs unrooted tree. 7

8. Neighbor Joining (Not assuming equal divergence)
Step by step summary:
Calculate all pairwise distances.
Pick two nodes (i and j) for which the relative distance is minimal (lowest).
Define a new node (x).
Calculate Dix and Djx - the distance of the chosen nodes I and J to the new node X, as well as the distance from X to all other nodes.
Continue until two nodes remain – connect with edge.

9. Step 1. Calculate all pairwise distances.B C D E E D C B A 41 39 22 - 43 20 18 10

10. Measuring Distance
Problem: unrelated sequences approach a fraction of difference expected by chance  The distance measure converges.
Jukes-Cantor

11. Measuring Distance (cont)
Euclidean Distance: Given a multiple sequence alignment, calculate the square root of the sum of the score at every position between two sequences
the score increases proportionally to the extent of dissimilarity between residues

12. Step 2. Pick two nodes (i and j) for which the relative distance is minimal (lowest).
Relative distance between i and j
Distance between i and j from the distance table
Negative values
As the average distance from the common ancestor to the rest of the nodes increases, Mij has a lower value.
Select pair that produce lowest value
Reevaluate M with every iteration
Distance of i from all other sequences
Number of leaves (=sequences) left in the tree

13. Step 2. Pick two nodes (i and j) for which the relative distance is minimal (lowest).B A 41 39 22 - 43 20 18 10

14. Step 2. Pick two nodes (i and j) for which the relative distance is minimal (lowest).
Etc. E D C B A
-44
-47.3
-74 -
-57.3
-64
A,B is the pair with the minimal Mi,j distance.
The Mij Table is used only to choose the closest pairs (lowest
value) and not for calculating the distances

15. Step 3. Define a new node (x)B C D E X

16. Step 4. Calculate Dix and Djx - the distance of the chosen nodes I and J to the new node X, as well as the distance from X to all other nodes.
Now we’ll calculate the distance from X to all other nodes. E D C X 31 29 - 20 18 10

17. Step 5 - Continue until two nodes remainX
-44
-49 -
New Mi,j table A B C D E X Y

18. E D Y 11 9 - 10
New Di,j table
Only 2 nodes are left. Let’s calculate all the distances to Z A B C D E X Y Z

19. And in newick tree format
The tree 6 4 E D C 5 9 12 10 B A 20 Z Y X
And in newick tree format
((C(D,E))(A,B))

20. ClustalW - Input Input sequences Scoring matrix Gap scoring
Input sequences
Scoring matrix
Gap scoring
Output format
address

21. Match strength in decreasing order: * : .
ClustalW - Output
Match strength in decreasing order: * : .

22. ClustalW - Output

23. ClustalW - Output

24. ClustalW - Output

25. Pairwise alignment scores
ClustalW - Output
Pairwise alignment scores
Building tree
Building alignment
Final score

26. ClustalW - Output

27. Match strength in decreasing order: * : .
ClustalW Output
Sequence names
Sequence positions
Match strength in decreasing order: * : .

28. ClustalW - Output

29. ClustalW - Output
Branch length

30. ClustalW - Output

31. ClustalW - Output