SMPD 287 Spring 2015 Bioinformatics in Medical Product Development. Final Examination

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1 Final Examination You have a choice between A, B, or C. Please your solutions, as a pdf attachment, by May 13, In the subject of the , please use the following format: firstname_lastname_x were X is either A, B, or C, depending on your choice. [A] From: Discovering Genomics, Proteomics, & Bioinformatics Chapter One, Section 1.1: First Patients Objectives: 1) Discover the complexity of apparently simple genetic diseases. 2) Use online bioinformatics tools to uncover new information. 3) Visualize protein connections and 3D structures. You are going to answer discovery questions 1 to 14 from Section One of What is wrong with my child? Skip the Math Minute 1.1, pages 7 and 8. Problem 1 Phase I: Clinical Presentation and Phase II: Family Pedigree DQ1: Spend about 5 to 10 minutes, as mentioned in the book. Not more. DQ2: To get to Genes and Disease From NCBI s main page Click on Genes & Expression under Resource List (A-Z) on the left hand side of the page Scroll down and click on Genes and Disease (By performing the following we can get to DMD: Click on Muscle and Bone from the new page Scroll down and click on Duchenne muscular dystrophy ) Alternatively: Go to wps.aw.com/bc_campbell_genomics_2/ Click on Genes and Diseases under Links DQ3: Do not spend more than 10 minutes, as mentioned in the book. DQ4: Do not spend more than 10 minutes, as mentioned in the book. Terms to know: histology Problem 2 Phase III: Karyotyping and Linkage Analysis Answer discovery questions 5 to 8. Terms to know: RFLP markers 2015 Sami Khuri 1

2 Problem 3 Phase IV: DNA Sequence Analysis Answer discovery questions 9 to 12. For discovery questions 10, 11, and 12, follow the instructions given in the hand-out. DQ10: Go to wps.aw.com/bc_campbell_genomics_2/ Click on locus of interest under Data From the new page Genetic Disease Sequences, scroll down and copy the sequence found under: Sequence which was conserved in all family members : NICKECPIIGFRYRSLKHFNYDICQSCFF. Go to the BLAST webpage at NCBI Click on protein blast under Basic BLAST Paste the sequence in the window Scroll down and choose Show results in a new window Click on the blue BLAST button. Inspect the top 20 to 25 hits and the bottom 20 to 25 hits. a) Do most of the top hits yield dystrophin in different species? Is the e-value significant? b) Do most of the bottom hits yield utrophin in different species? Is the e-value significant? c) What is the accession number of the top hit? When was it last updated? DQ11: Go to wps.aw.com/bc_campbell_genomics_2/ Click on PDB under Links. Alternatively, go to Enter the code 1dxx and click on the search button. Click on Protein Workshop (on the right hand side of the page). From the new page with title: RSCB PDB Protein Workshop (powdered by the MBT): 1DXX, click on the Visibility button. Click on the small + sign next to Chain A to view the amino acids with their positions. Scroll to 54 LEU and click on it. This will put a small mark on the graph. You might have to rotate the molecule with the mouse to be able to see the position of leucine on the graph. Recall that instead of Leucine (in position 54), BB and GG have Arginine (see Table 1.1). In what kind of secondary structure of the protein does this mutation lie? Now choose Labels from Select your tool Choose Label by residues from Change the tool s options, if necessary Sami Khuri 2

3 Click once more on 54 Leu to see the position of leucine on the molecule. DQ12: In this exercise, we will do the same as DQ11, except that we will highlight the sequence DVQKKTFTKW from position 15 to 24, instead of just amino acid leucine in position 54. We will use two methods: Protein Workshop and Sequence details. A) Protein Workshop Go to wps.aw.com/bc_campbell_genomics_2/ Click on locus of interest under Data From the new page Genetic Disease Sequences, scroll down and copy the sequence found under: Find this sequence. Go once again to wps.aw.com/bc_campbell_genomics_2/ Click on PDB under Links. Alternatively, go to Enter the code 1dxx and click on the Search button. Click on Protein Workshop (on the right-hand side of the page). From the new page with title: PDB Protein Workshop (powdered by the MBT): 1DXX, click on the Visibility button. Click on the small + sign next to Chain A to view the amino acids with their positions. Scroll to 15 ASP click on it, push the shift key and click on 24 TRP. This will choose all the amino acids from position 15 to 24 and will put small marks on the graph. You might have to rotate the molecule with mouse to be able to see the position of the 10 amino acids on the graph. a) In what kind of secondary structure of the protein does this mutation lie? Go once again to wps.aw.com/bc_campbell_genomics_2/ Click on PDB under Links. Alternatively, go to Enter the code 1dxx and click on the search button. From the new page, click on Sequence tab from the menu bar in the upper half of the page. Scroll down and inspect the sequence. b) What kind of secondary structure of the protein does the peptide help form? Problem 4 Answer discovery questions 13 and 14. DQ13: Do not spend too much time on this question. Just try to formulate an educated guess. DQ14: Do not spend too much time on this question. Just try to formulate an educated guess. Terms to know: dystrophin 2015 Sami Khuri 3

4 Chapter One, Section 1.2: The Next Step in Understanding the Disease - Continuation Objectives: 1) Discover the complexity of apparently simple genetic diseases. 2) Use online bioinformatics tools to uncover new information. 3) Visualize protein connections and 3D structures. 4) Understand the value of a model research organism. 5) Recognize diagrams of interacting components as circuit diagrams. You are going to answer discovery questions 24 to 32 from Section Two of What is wrong with my child? Read pages 13 to 20 (up to DQ 32) and answer the discovery questions as you go along. Skip Math Minute 1.3. Problem 5 Solve Discovery Questions 24 to 29. DQ28: Go to NCBI at: Select Protein from the Search drop-down menu. Enter the word sarcoglycan and hit the blue Search button to search the protein database. From the new page, you will see Homo sapiens (113) on the right hand side of the page. Click on (113) to get the human sarcoglycans. How many different human sarcoglycan genes are there? You know there are at least four, but can you find more sarcoglycans? Terms to know: penetrance Problem 6 Solve Discovery Questions 30, 31, and 32. DQ32: Follow the instructions in the textbook. If you want to narrow down the number of hits, try dystrophy* AND signal. Terms to know: consanguineous, founder effect, digenic Sami Khuri 4

5 [B] Thalassemia Beta Thalassemia by Antonio Cao and Renzo Galanello, was published in Genetics in Medicine in 2010 (vol. 12, issue 2, pages 61-76). 1) Explain in your own words Figure 1 and Figure 2 one paragraph for each figure. 2) Explain in your own words Figure 4. Fully explain every single part of Fig 4. 3) Figure 3 of the article gives the mutations of the β globin gene associated with the phenotypes in the β-thalassemias in the Mediterranean Region and in the Asian Region. Choose 3 mutations from each region (6 mutations in all) that were not covered in the class, and for each mutation (as was done in the course): a) locate the mutation on the sequence, b) explain what consequences on the protein it might have (as was done in the course). 4) Recall the upper half of Table 1 of Changes in the Epidemiology of Thalassemia in North America: A New Minority Disease by Elliott P. Vichinsky, et al. we studied in class. Where do you think the majority of the patients reported in Table 1 originally come from? [C] Origin of HIV We are going to build phylogenetic trees with 12 sequences from HIV1, HIV2 and SIV. The sequences are from NCBI. The table lists, from left to right: Num: the number representing the order in which the sequences were obtained from NCBI Isolate: the isolate names of the genomes as given by NCBI Accession Number: GenBank accession numbers of the whole genomes Length in base pairs: the length of the genome Posted Entry Date: the date found in the corresponding database entry Organism: the organism to which the genome belongs. Num Isolate Accession Length in Posted Entry Date Organism Number base pairs 1 HIV1_ELI K FEB-2002 Human 2 HIV1_BRU K AUG-1993 Human 3 HIV1_MAL X APR-2005 Human 4 HIV1_NDK M AUG-1993 Human 5 HIV2_D205 X NOV-2006 Human 6 HIV2_ROD M MAY-1996 Human 7 HIV2_ST M MAY-1996 Human 8 HIV2_UCI L AUG-1993 Human 9 SIV_Mm251 M NOV-2000 Macaque 10 SIV_CPZ X APR-2005 Chimpanzee 11 SIV_AGM M MAR-2006 Af. green monkey 12 SIV_SMM X APR-2005 Sooty mangabey mk Sami Khuri 5

6 Three files, env_protein_sequences.txt, gag_protein_sequences.txt, and pol_protein_sequences.txt containing the amino acid sequences for the env, gag and pol proteins from the 12 isolates were created. In other words: o env_protein_sequences.txt contains 12 env protein sequences, one from each of the 12 genomes found in the table. o gag_protein_sequences.txt contains 12 gag protein sequences, one from each of the 12 genomes found in the table. o pol_protein_sequences.txt contains 12 pol protein sequences, one from each of the 12 genomes found in the table. Part A: Retrieve env_protein_sequences.txt and copy all 12 sequences. Go to and paste all 12 sequences in the window. Click on More options under STEP 2 and open the dropdown window under OUTPUT FORMAT. Choose PHYLIP from the pull-down menu. We need to have the resulting alignment in the phylip format for subsequent steps. Click on Submit of STEP 3 to align the 12 env protein sequences and wait for the result. The new page contains the actual alignment. The multiple sequence alignment is preceded by 2 numbers: o The first number represents the number of sequences (rows). In our example: 12. o The second represents the total number of columns in the alignment. In our example: 950. Use Download Alignment File to save the alignment under clustaloenv.txt on your desktop. In a new window: Go We are going to use the PHYLIP package to construct phylogenetic trees. Click on phylogeny under Programs Click on distance and then on protdist. The protdist program from the PHYLIP package will compute the pairwise distances of the 12 sequences from the multiple sequence alignment Sami Khuri 6

7 Copy the entire content of the clustalo-env.txt you obtained above, including the first row with the two numbers, and paste it in the Phylip protdist window. o Alternatively, you can choose the upload button under Alignment File by clicking on it and then clicking on Browse to upload clustalo-env.txt. Then choose clustalo-env.txt from the drop-down window next to select and click on select. The contents of clustalo-env.txt should appear in the display window. Enter your address in the appropriate window, after clicking on set at the top of the PHYLIP page, on the right-hand side. Scroll down to Bootstrap options and choose Yes in the box to the right of Perform a bootstrap before analysis?. Type 1 in the window to the right of Random number seed (must be odd). The random seed is used to get randomness in the bootstrap algorithm. Keep the default value of 100 for the number of replicates. We want the package to calculate distance matrices for the 100 bootstrap replicates (the 100 problem instances that are randomly generated). Scroll up to the top of the page and click on the Run button. You might be prompted to validate your submission. Do it please. You will get a new page, and will have to wait (sometimes up to a few minutes) until you see an output file, protdist.outfile under Outfile (PhylipDistanceMatrix). You will also get messages from PHYLIP. The protdist.outfile file will be used as input file to PHYLIP s neighbor. Go to the row right underneath the protdist.outfile window (that starts with full screen ) and choose neighbor (inflile) from the drop-down window right before further analysis. Click on further analysis to obtain a new page. From the new page, click on advanced options Scroll down to Bootstrap options : o Choose Yes for Analyze multiple data sets (M) o Type 100 for How many data sets 2015 Sami Khuri 7

8 o Type 1 in the Random number seed for multiple dataset (must be odd) window o Choose Yes for Computer a consensus tree Scroll back up and click on Run. From the new page, you will have several different output files under results. We are interested in the very first one: consense.outfile. If you scroll down in that window, you will see the consensus tree produced by the 100 bootstrap runs. We would like to save consensus.outfile (in a readable format). Click on full screen right underneath the Consense output file Save the resulting page under env consensus Open the file you have just saved: env_consensus_outfile, and scroll down to see the consensus tree with the bootstrap values for each branch. Part B: 1) Repeat the procedure described in part A with gag_hiv_siv_sequences.txt and save the resulting consensus trees under gag_consense_outfile.txt. 2) Repeat the procedure described in part A with pol_hiv_siv_sequences.txt and save the resulting consensus trees under pol_consense_outfile.txt. Part C: Study carefully the three consensus trees and answer the following questions: 1) What do the trees show with regards to the HIV and SIV relationships? 2) Why do SIV s cluster with both HIV-1 and HIV-2? 3) Which HIV type, HIV-1 or HIV-2, is more closely related to the SIV from the sooty mangabey? Which type is more closely related to the SIV from the chimpanzee? What does this tell you about the origin of HIV-1 and HIV-2? This project was adapted from The Origin and Evolution of HIV from Siv Andersson s laboratory at Uppsala University, Sweden Sami Khuri 8