Introduction to Bioinformatics

1. Introduction to Bioinformatics Tutorial 4 Multiple Alignment and Phylogeny

2. ClustalW Input Alignment format Fast alignment? Fast alignment options Scoring matrix Gap scoring Input sequences Phylogenetic trees

3. ClustalW Output (1) Input sequences Pairwise alignment scores Building alignment Final score

4. ClustalW Output (2) Sequence names Sequence positions Match strength in decreasing order: * : .

5. Distance representation chimp monkey human Phylogenetic Trees • Represent closeness between many entities • In our case, genomic or protein sequences Unobserved commonality Observed entity

6. Rooting Trees • A tree can be hung from a root • Adds directional information • Requires addition of ‘outgroup’ So we hang the tree from where it joins We know this is furthest pig monkey human chimp

7. Speciation Number of mutations Phylogeny and Evolution Common Ancestor Evolutionary Time

8. Tree Reconstruction • Build tree based on organism sequences • Distance-based methods • Use pairwise alignment scores to build tree • Ignores sequences after initial alignments • Character-based methods • Learn a tree with intermediate sequences that minimizes total number of mutations • Slower but generally better results

9. 1 2 3 4 Distance-based Example (1)

10. Distance-based Example (2) 1 2 3 4

11. Distance-based Example (3) 3 4 1 2

12. Newick Tree Format (CFTR_SHEEP:0.01457, (CFTR_HUMAN:0.16153, (CFTR_MOUSE:0.70599, (CFTR_RABIT:2.76042, (CFTR_SQUAC:1.27192, CFTR_XENLA:0.28818) :3.42183) :0.77076) :0.65873) :0.73937, CFTR_BOVIN:0.00953);

13. Phylodendron Input Graphical style Newick tree description Tree size Orientation

14. Calculation of HIV/SIV Neighbor-joining tree Why phylogenetic analyses? Mutations accumulate in the genomes of pathogens, especially viruses, during a spread of an infection. This can be used to document the history of transmission events. Phylogenetic analysis of these mutations may not only be used to reconstruct the history of a pathogen's spread through host populations but can also be used to make predictions about it's future progress. The unsolved HIV/SIV relationshipOne interesting case, where phylogenetic treebuilding is useful, is the unsolved HIV/SIV relationship: HIV-1, HIV-2 and SIV.AIDS (acquired immunodeficiency syndrome) is caused by two different human viruses: HIV-1, group M and O HIV-2, subtypes A to E There are many related viruses in a variety of non-human primates. These related viruses are called SIV (simian immunodeficiency viruses).

15. Calculation of HIV/SIV Neighbor-joining tree • Phylogenetic studies have shown that primate lentiviruses are all in the same clade. Within this clade there are five major lineages (the subscripts denotes the host) : • HIV-1 and SIVCPZ (Chimpanzee) • HIV-2, SIVSM (Sooty mangabey) and SIVMAC (Captive macaque) • SIVAGM (African green monkey) • SIVMND (Mandrill) • SIVSYK (Sykes´ monkey) The NJ tree in our example is based on the poly protein sequence from HIV-1, HIV-2 and SIV with HTLV-1 as an outgroup. HTLV-1 (human T-lymphotropic virus type 1) is another human retroviral pathogen that has originated from related simian viruses.

16. Calculation of HIV/SIV Neighbor-joining tree • Step by step summary: • Define all taxa and calculate all pairwise distances. • Pick two nodes in the star (i and j) for which the distance is minimal. • Define a new node (x) and calculate ri and rj. • Calculate dix and djx, thereby joining x to i and j respectively. • Remove i and j from the star and insert x instead. • Calculate dxm for all m in the star. • Continue until the star has been resolved and root the tree in a final step.

17. minimum Step1

18. Step1(cont.) The calculation starts with the star: The branch lengths between node 5 and 10 and between 6 and 10 are calculated with these formulas: In this case L = 9 New node x = 10 ri=r5=Σd5k/(L-2) = 3.22406/(9-2) = 0.46058 rj=r6=Σd6k/(L-2) = 3.22758/(9-2) = 0.461083 dix=d510=(d5 6 + r5 - r6)/2 = (0.06088 + 0.46058 - 0.461083)/2 = 0.0301886 djx=d6 10 = d5 6 - d5 10 = 0.06088 - 0.0301886 = 0.0306914

19. Step1(cont.)

20. minimum Step2

21. Step2(cont.) Calculation of the new branches: In this case L = 8 New node x = 11 ri=r3=Σd3k/(L-2) = 2.715455/(8-2) = 0.452576 rj= r4=Σd4k/(L-2) = 2.50096/(8-2)=0.416827 dix=d3 11=(d3 4 + r3 - r4)/2 = (0.125 + 0.452576 - 0.416827)/2 = 0.080375 djx=d4 11 = d3 4 - d3 11 = 0.125 - 0.080375 = 0.044625

22. Step1(cont.)

23. minimum Step3

24. Step3 (cont.) Calculation of the new branches: In this case L = 7 New node x = 12ri=r2=Σd2k/(L-2) = 2.252265/(7-2) = 0.450453 rj=r11=Σd11k/(L-2) = 2.108208/(7-2)=0.4216415 dix=d212=(d211 + r2 - r11)/2 = (0.109705 + 0.450453 - 0.4216415)/2 = 0.069258 djx=d1112 = d211 - d212 = 0.109705 - 0.069258 = 0.040447

25. Step1(cont.)

26. Step 7 In this case L = 3 New node x = 16:r13= 0.843684 r15=0.728574 d1316 = 0.131758 d1516 = 0.016648

27. Step 7 (cont.) Because node 9 is the outgroup, the root will be placed between node 9 and the other nodes. The distance between node 9 and the first internal node is 0.563519.

28. This means that HIV-1 and HIV-2 have originated independently from two different SIV strains. Conclusions HIV-1 seems to be more closely related to SIV from chimpanzee. There also seems to have been a cross-species transmission from human to MAN/MAC. There must have been a cross-species transmission from chimpanzee SIV to human HIV-1. HIV-2 (H2) is more closely related to SIV (S) from sooty mangabey than to HIV-1 (H1).

29. Conclusions As one can see the branch between the H2-ROD A and the to SIV taxa has a low support. Only 56% of the trees have this topology. Therefore the transmission events from human to non-human primates are very uncertain.

30. Exercise In this exercise you will perform a phylogenetic analysis of the human globin sequences. You will compare your results to current prevalent knowledge on the globin family, according to the following summary on the globin sequences: Myoglobin and hemoglobins diverged from one another before the emergence of worms, about 800 million year ago. The hemoglobins diverged into two families (the α-family and β-family) following a gene duplication, about 450 million years ago, which is before the emergence of mammals. The α-family diverged into the zeta, teta and alpha genes, and the β-family diverged into the beta, gamma_G, gamma_A, delta and epsilon genes, all following a series of gene duplications. The most recent duplication was that gamma_G from gamma_A, which occurred around the separation of the simians (humans, chimp, gorilla, etc.) from the pro-siminas (such as lemurs and lorises), about 55 million years ago. (adapted from Graur and Li, 1999)

31. Exercise (cont.) • Reconstruct the phylogenetic tree of the human globins using Neighbor joining. Make sure tree is properly rooted (by defining an outgroup) according to the information in the above summary. Point out where the hemoglobins and myoglobin diverged, and where the α-family and β-family diverged. • Which of the following groups are monophyletic according to the tree you obtained: • (i) alpha, beta, delta, • (ii) alpha, teta, zeta, (iii) epsilon, beta, delta • Bootstrap the tree you built with 1000 bootstrap iterations. Display the tree with the bootstrap values displayed. On which branch was the lowest bootstrap value obtained? Explain what this means.