BIOL398-05/S17:Week 12: Difference between revisions
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#** Select the area of the entire adjacency matrix. | #** Select the area of the entire adjacency matrix. | ||
#** Click the Data tab and click the custom sort button. | #** Click the Data tab and click the custom sort button. | ||
#** Sort Column A alphabetically, being sure to exclude the header row. | |||
#** Now sort row 1 from left to right, excluding cell A1. In the Custom Sort window, click on the options button and select sort left to right, excluding column 1. | |||
#* Name the worksheet containing your organized adjacency matrix "network" and Save. | |||
# Now we will visualize what these gene regulatory networks look like with the GRNsight software. | # Now we will visualize what these gene regulatory networks look like with the GRNsight software. | ||
#* Go to the [http://dondi.github.io/GRNsight/ GRNsight] home page | #* Go to the [http://dondi.github.io/GRNsight/ GRNsight] home page. | ||
#* Select the menu item File > Open and select | #* Select the menu item File > Open and select the regulation matrix .xlsx file that has the "network" worksheet in it that you formatted above. If the file has been formatted properly, GRNsight should automatically create a graph of your network. Move the nodes (genes) around until you get a layout that you like and take a screenshot of the results. Paste it into your PowerPoint presentation. | ||
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Revision as of 09:28, 6 April 2017
This page is currently under construction.
This journal entry is due on Thursday, April 20 at midnight PDT (Wednesday night/Thursday morning). NOTE that the server records the time as Eastern Daylight Time (EDT). Therefore, midnight will register as 03:00. NOTE also that the assignment is due the week after Easter Break.
Individual Journal Assignment
- Store this journal entry as "username Week 12" (i.e., this is the text to place between the square brackets when you link to this page).
- Create the following set of links. (HINT: These links should all be in your personal template that you created for the Week 1 Assignment; you should then simply invoke your template on each new journal entry.)
- Link to your journal entry from your user page.
- Link back from your journal entry to your user page.
- Link to this assignment from your journal entry.
- Don't forget to add the "BIOL398-05/S17" category to the end of your wiki page.
Homework Partners
Please meet with your partner (either face-to-face or virtually) at least once when preparing this assignment. Even though you may work together to understand the article, your journal assignment must be completed individually. It is not acceptable to do a joint assignment and copy it over to each others' journal page.
- Lauren, Cameron
- Nika, Conor
- Maggie
Electronic Lab Notebook
Complete your electronic notebook that gives the details of what you did for the assignment this week. Your notebook entry should contain:
- The purpose: what was the scientific purpose of your investigations?
- Note that this is different than the learning purpose.
- Your workflow or methods: what did you actually do? Give a step by step account.
- There should be enough detail provided so that you or another person could re-do it based solely on your notebook.
- You may copy protocol instructions to your page and modify them as to what you actually did, as long as you provide appropriate attribution in the acknowledgments and references section.
- Take advantage of the electronic nature of the notebook by providing screenshots, links to web pages, etc.
- Your results: the answers to the questions in the protocol, plus any other results you gathered. Your results will include some or all of the following: images, plots, data, and files.
- Note that files left on the Desktop or My Documents or Downloads folders on the Seaver 120 computers will be deleted upon restart of the computers. Files stored on the
T:
drive will be saved. However, it is not a good idea to trust that they will be there when you next use the computer. - Thus, it is a critical skill for data and computer literacy to back-up your data and files in at least two ways:
- Upload the files to this wiki.
- Upload the files to Box.
- Back them up on your personal flash drive.
- References to data and files should be made within the methods and results section of your notebook, listed above.
- In addition to these inline links, create a Data and Files section of your notebook to make a list of the files generated in this exercise.
- Note that files left on the Desktop or My Documents or Downloads folders on the Seaver 120 computers will be deleted upon restart of the computers. Files stored on the
- A scientific conclusion: what was your main finding for today's project? Did you fulfill the purpose? Why or why not?
- The Acknowledgments section, see below.
- The References section, see below.
Acknowledgments
In this section, you need to acknowledge anyone who assisted you with your assignment, either in person, electronically, or even anonymously without their knowledge.
- You must acknowledge your homework partner or team members with whom you worked, giving details of the nature of the collaboration. You should include when and how you met and what content you worked on together. An appropriate statement could be (but is not limited to) the following:
- I worked with my homework partner (give name and link name to their user page) in class. We met face-to-face one time outside of class. We texted/e-mailed/chatted online three times. We worked on the <details> portion of the assignment together.
- Acknowledge anyone else you worked with who was not your assigned partner. This could be Dr. Dahlquist or Dr. Fitzpatrick (for example, via office hours), the TA, other students in the class, or even other students or faculty outside of the class.
- If you copied
wiki syntax
or a particular style from another wiki page, acknowledge that here. Provide the user name of the original page, if possible, and provide a link to the page from which you copied the syntax or style. - If you need to reference content (such as the methods of a protocol) also acknowledge it here and include a formal citation in your References section (see below).
- You must also include this statement unless otherwise noted:
- "Except for what is noted above, this individual journal entry was completed by me and not copied from another source."
- Sign your Acknowledgments section with your wiki signature.
References
- In this section, you need to provide properly formatted citations to any content that was not entirely of your own devising. This includes, but is not limited to:
- methods
- data
- facts
- images
- documents, including the scientific literature
- The references in this section should be accompanied by in text citations on your page that refer to these references.
- Do not include citations/references to sources that you did not use.
- Generally, you should include a reference to that week's assignment page.
- The references should be formatted according to the APA guidelines.
- For more detailed instructions on how to cite journal articles, books, or web pages, please see the document Guidelines for Literature Citations in a Scientific Paper that you were given on the first day of class.
Microarray Data Analysis
For this assignment, we will continue to analyze the data from the Week 11 assignment. Whatever you did not finish for Week 11 gets rolled into this assignment with the additions below.
Background
This is a list of steps required to analyze DNA microarray data.
- Quantitate the fluorescence signal in each spot
- Calculate the ratio of red/green fluorescence
- Log2 transform the ratios
- Steps 1-3 have been performed for you by the GenePix Pro software (which runs the microarray scanner).
- Normalize the ratios on each microarray slide
- Normalize the ratios for a set of slides in an experiment
- Steps 4-5 was performed for you using a script in R, a statistics package (see: Microarray Data Analysis Workflow)
- You will perform the following steps:
- Perform statistical analysis on the ratios
- Compare individual genes with known data
- Steps 6-7 are performed in Microsoft Excel
- Pattern finding algorithms (clustering)
- Map onto biological pathways
- We will use software called STEM for the clustering and mapping
- Identifying regulatory transcription factors responsible for observed changes in gene expression
- Dynamical systems modeling of the gene regulatory network
- The modeling will be performed in MATLAB
For the modeling project, each pair of students will analyze a Dahlquist lab microarray dataset from a particular strain of yeast.
- Wild type: Maggie
- Δgln3: Nika and Conor
- Δhap4: Lauren and Cameron
You will download your assigned Excel spreadsheet from Box. You were e-mailed a link to do this before class. Because the Dahlquist Lab data is unpublished, please do not post it on this public wiki. Instead, post the file(s) back to Box, which is protected by a password.
Before you begin...
Remember to set your computer to display file extensions and your browser to download files to a folder you specify. Instructions are on the Week 11 Assignment page.
Using YEASTRACT to Infer which Transcription Factors Regulate a Cluster of Genes
In the previous analysis using STEM, we found a number of gene expression profiles (aka clusters) which grouped genes based on similarity of gene expression changes over time. The implication is that these genes share the same expression pattern because they are regulated by the same (or the same set) of transcription factors. We will explore this using the YEASTRACT database.
- Open the gene list in Excel for the one of the significant profiles from your stem analysis. Choose a cluster with a clear cold shock/recovery up/down or down/up pattern. You should also choose one of the largest clusters.
- Copy the list of gene IDs onto your clipboard.
- Launch a web browser and go to the YEASTRACT database.
- On the left panel of the window, click on the link to Rank by TF.
- Paste your list of genes from your cluster into the box labeled ORFs/Genes.
- Check the box for Check for all TFs.
- Accept the defaults for the Regulations Filter (Documented, DNA binding plus expression evidence)
- Do not apply a filter for "Filter Documented Regulations by environmental condition".
- Rank genes by TF using: The % of genes in the list and in YEASTRACT regulated by each TF.
- Click the Search button.
- Answer the following questions:
- In the results window that appears, the p values colored green are considered "significant", the ones colored yellow are considered "borderline significant" and the ones colored pink are considered "not significant". How many transcription factors are green or "significant"?
- Copy the table of results from the web page and paste it into a new Excel workbook to preserve the results
- Are GLN3 or HAP4 on the list? If so, what is their "% in user set", "% in YEASTRACT", and "p value".
- For the mathematical model that we will build, we need to define a gene regulatory network of transcription factors that regulate other transcription factors. We can use YEASTRACT to assist us with creating the network. We want to generate a network with approximately 15-30 transcription factors in it.
- You need to select from this list of "significant" transcription factors, which ones you will use to run the model. You will use these transcription factors and add GLN3 and HAP4 if they are not in your list. Explain in your electronic notebook how you decided on which transcription factors to include. Record the list and your justification in your electronic lab notebook.
- Go back to the YEASTRACT database and follow the link to Generate Regulation Matrix.
- Copy and paste the list of transcription factors you identified (plus HAP4 and GLN3) into both the "Transcription factors" field and the "Target ORF/Genes" field.
- We are going to use the "Regulations Filter" options of "Documented", "Only DNA binding evidence"
- Click the "Generate" button.
- In the results window that appears, click on the link to the "Regulation matrix (Semicolon Separated Values (CSV) file)" that appears and save it to your Desktop. Rename this file with a meaningful name so that you can distinguish it from the other files you will generate.
Visualizing Your Gene Regulatory Networks with GRNsight
We will analyze the regulatory matrix files you generated above in Microsoft Excel and visualize them using GRNsight to determine which one will be appropriate to pursue further in the modeling.
- First we need to properly format the output files from YEASTRACT. You will repeat these steps for each of the three files you generated above.
- Open the file in Excel. It will not open properly in Excel because a semicolon was used as the column delimiter instead of a comma. To fix this, Select the entire Column A. Then go to the "Data" tab and select "Text to columns". In the Wizard that appears, select "Delimited" and click "Next". In the next window, select "Semicolon", and click "Next". In the next window, leave the data format at "General", and click "Finish". This should now look like a table with the names of the transcription factors across the top and down the first column and all of the zeros and ones distributed throughout the rows and columns. This is called an "adjacency matrix." If there is a "1" in the cell, that means there is a connection between the trancription factor in that row with that column.
- Save this file in Microsoft Excel workbook format (.xlsx).
- Check to see that all of the transcription factors in the matrix are connected to at least one of the other transcription factors by making sure that there is at least one "1" in a row or column for that transcription factor. If a factor is not connected to any other factor, delete its row and column from the matrix. Make sure that you still have somewhere between 15 and 30 transcription factors in your network after this pruning.
- Only delete the transcription factor if there are all zeros in its column AND all zeros in its row. You may find visualizing the matrix in GRNsight (below) can help you find these easily.
- For this adjacency matrix to be usable in GRNmap (the modeling software) and GRNsight (the visualization software), we need to transpose the matrix. Insert a new worksheet into your Excel file and name it "network". Go back to the previous sheet and select the entire matrix and copy it. Go to you new worksheet and click on the A1 cell in the upper left. Select "Paste special" from the "Home" tab. In the window that appears, check the box for "Transpose". This will paste your data with the columns transposed to rows and vice versa. This is necessary because we want the transcription factors that are the "regulatORS" across the top and the "regulatEES" along the side.
- The labels for the genes in the columns and rows need to match. Thus, delete the "p" from each of the gene names in the columns. Adjust the case of the labels to make them all upper case.
- In cell A1, copy and paste the text "rows genes affected/cols genes controlling".
- Finally, for ease of working with the adjacency matrix in Excel, we want to alphabatize the gene labels both across the top and side.
- Select the area of the entire adjacency matrix.
- Click the Data tab and click the custom sort button.
- Sort Column A alphabetically, being sure to exclude the header row.
- Now sort row 1 from left to right, excluding cell A1. In the Custom Sort window, click on the options button and select sort left to right, excluding column 1.
- Name the worksheet containing your organized adjacency matrix "network" and Save.
- Now we will visualize what these gene regulatory networks look like with the GRNsight software.
- Go to the GRNsight home page.
- Select the menu item File > Open and select the regulation matrix .xlsx file that has the "network" worksheet in it that you formatted above. If the file has been formatted properly, GRNsight should automatically create a graph of your network. Move the nodes (genes) around until you get a layout that you like and take a screenshot of the results. Paste it into your PowerPoint presentation.