Simulation of Molecular Evolution with Bioinformatics Analysis
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1 Simulation of Molecular Evolution with Bioinformatics Analysis Barbara N. Beck, Rochester Community and Technical College, Rochester, MN Project created by: Barbara N. Beck, Ph.D., Rochester Community and Technical College, Rochester, MN Chi-Cheng Lin, Ph.D., Winona State University, Winona, MN Mingrui Zhang, Ph.D., Winona State University, Winona, MN Gayle Olsen, M.S., C.N.P., Winona State University, Winona, MN Robyn L. Keyport, M.Ed., Hastings High School, Hastings, MN 1
2 Learning objective Students will cause a set of molecules to evolve and then use bioinformatics computational tools to analyze the relatedness of this set of molecules, displaying the results as phylogenetic trees. Students will gain an understanding of how phylogenetic trees display evolutionary relationships. The evolved set of molecules is created by manipulating strings of Pop-It beads consisting of four colors of beads, representing the four DNA bases. Base substitutions result from substituting one color of bead for another. 2
3 The ancestor (original sequence) molecule diverges into two lineages, each of which undergoes an independent mutation. Each of these lineages also diverges, with the descendents undergoing independent mutations until a population of eight lineages is created. #1 #2 #1a #1b #2a #2b #1aa #1ab _ #1ba #1bb #2aa #2ab _ #2ba #2bb = a mutated site Data recording Line up bead strings with bead-size Excel spreadsheet pages (cells in spreadsheet same size as beads) to record mutations (changes in bead color). 3
4 Data recording Transfer changes to live spreadsheet. Data recording Save the altered file with a new name and then select the datacontaining rows 3-10 from column B to BG and click Copy. 4
5 Data format conversion We need to convert the bead data from RGBY (bead colors) to ACGT (nucleotides) and to convert it to FASTA format, a format compatible with the publicly available analysis tools. Open a new document in Wordpad or Notepad (easier) and click Paste to transfer your data to one of these text editors. Data format conversion Click File, Save as, choose a location for storing the file (the Desktop is easy), type in a filename, and in the Save as type field, choose Text Document (*.txt). 5
6 Data format conversion Open the Java file BBead.jar by double-clicking it. Browse for the data file you just saved (as a.txt file) and select it as the input file. Data format conversion Type in an output file name and then click Convert Sequence. The converted sequence will be displayed on the screen and the output file will be created in the location of the BBead.jar file. 6
7 Open a web browser (Explorer, Firefox) and enter the URL A new user is required to register for a FREE account and then log in. We will upload our FASTA-formatted data file and use the computational tools available through this site to align the sequences and calculate the phylogenetic tree. Your work will be saved in your session. 7
8 To start a new session, click on Session Tools. You will then see the following screen. Highlight Start New Session and click Run 8
9 Name your session and then click Start New Session. Your new session will be highlighted as shown below. Our data are DNA sequences, so we will use Nucleic Tools. Click on Nucleic Tools. You will see the following page. -Empty means that no DNA sequences have yet been imported. Click Add New Nucleic Sequence and then click Run. 9
10 Click on Browse, find the.txt data file that you converted to FASTA format, select it, and then click Upload File. Once the file is uploaded, your sequences will appear on the page. Click Save to store them on the site. 10
11 In the drop-down list, first highlight Select All Sequences (all the boxes should get checked), and then highlight CLUSTALW Multiple Sequence Alignment. CLUSTALW compares and then aligns the sequences. You are now on a Check page. Verify that all your sequences are listed. (If not, click Abort and re-do the last step.) Then click Submit. Click on Submit 11
12 The alignment may take a minute or so. When it is finished, scroll down the page. The sequence alignments are color-coded. Blue indicates that all the nucleotides at that position are identical. Guide trees vs. phylogenetic trees If you scroll down below the alignment, you will see a guide tree displayed. CLUSTALW builds a guide tree to help align the sequences; the guide tree is not the same as a phylogenetic tree, although it may look very similar. In order to calculate the phylogenetic tree, we will do the following: On the CLUSTALW result page, click Import Alignment(s) near either the top or the bottom of the page. 12
13 Calculating phylogenetic trees On the result page, check the box in front of CLUSTALW - Nucleic. In the drop-down box, highlight and click DRAWTREE. Calculating phylogenetic trees Click Submit on the newly returned page and you will see the inferred unrooted phylogenetic tree in the result. An unrooted tree does not assume a direction of evolution and therefore will not include a single ancestor node, but will show evolutionary distances. Unrooted Phylogenetic Tree 13
14 Calculating Phylogenetic Trees Click Return to get back to the page showing the alignment. Highlight and click DRAWGRAM in the drop-down box. Click Submit on the newly returned page and you will see the inferred rooted tree in the result. The rooted tree assumes there is a direction of evolution and displays a single ancestor node. Rooted Phylogenetic Tree Tree analysis The detailed results obtained will vary each time, of course, since the input sequences are created by random mutation of the original sequence. However, the overall picture should be similar: The paired sequences, 1aa and 1ab, 1ba and 1bb, 2aa and 2ab, 2ba and 2bb should be most closely related to each other. Then the 1aa 1ab and 1ba 1bb pairs should cluster, as should the 2aa 2ab and 2ba 2bb pairs. The clustering reflects the temporal order in which the lineages were derived, thus showing that the alignment and tree-drawing algorithms can correctly infer the ancestral relationships. 14
15 Conclusions It is hoped that the hands-on activity of creating a set of related molecules and then of analyzing the sequence data represented by those molecules will give students a firmer grasp on the concept of how phylogenetic trees display information about evolutionary relationships. This activity can be extended by providing (or having students find) protein or DNA sequences using the Taxonomy Browser tool at the NCBI Taxonomy Homepage and then using the tools at the SDSC Biology Workbench to align the sequences and draw the phylogenetic trees. All files for this exercise, and a pdf version of this presentation are available by from the presenter at barbara.beck@roch.edu 15
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