Difference between revisions of "Dbashour Week 14"

From LMU BioDB 2017
Jump to: navigation, search
(completed week 10 assignment)
 
(deliverables updated)
Line 95: Line 95:
 
#* each of the genelist and GOlist files for each of your significant profiles.
 
#* each of the genelist and GOlist files for each of your significant profiles.
 
# Write a paragraph-length conclusion for this week's exercise.
 
# Write a paragraph-length conclusion for this week's exercise.
 +
 +
= Deliverables =
 +
[[File:Yeastract_Matrix_DB]]

Revision as of 22:42, 7 December 2017

Electronic Notebook

Week 8 Corrections

Week 10 Corrections

Week 10 Continued

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.

  1. I opened the gene list in Excel for profile 45 of my stem analysis. I chose this cluster because it had a cold shock/recovery up/down or down/up pattern and it was one of the largest clusters.
    • I then copied the list of gene IDs onto my clipboard.
  2. I launched a web browser and went to the YEASTRACT database.
    • On the left panel of the window, I clicked on the link to Rank by TF.
    • I pasted my list of genes from my chosen 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.
  3. 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"?
    • There are 30 green or significant transcription factors.
      • I copied the table of results from the web page and pasted it into a new Excel workbook to preserve the results.
        • I upload the Excel file to Box and link to it below in the deliverables section of this wiki, naming it "Yeastract_Results_DB_Gene_hAPI.
        • My transcription factor is on the list. It's % in user set is 0.3085%, its % in yeastract is 0.1044%, and its p value is 1E-13.
  4. For the mathematical model and GRNsight, 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.
    • I chose all 30 of the significant transcription factors on my list, adding HAP4 since it was not already on the list. I chose these transcription factors among the rest because they are all significant so this way I can analyze all the significant TFs keeping in mind their % in user set, % in yeastract, and p value. All 30 TFs are listed below.
      • Sfp1p
      •  Msn2p
      • Yhp1p
      • Yox1p
      • Ace2p
      • Gln3p
      • Yap1p
      • Pdr3p
      • Ume6p
      •  Pdr1p
      •  Stb5p
      •  Swi5p
      •  YLR278C
      •  Mig2p
      •  Asg1p
      •  Tup1p
      •  Gcr2p
      •  Msn4p
      •  Rim101p
      •  Gcn4p
      • Sut1p
      • Mcm1p
      •  Met4p
      •  Rlm1p
      •  Ino4p
      •  Ndt80p
      •  Zap1p
      •  Abf1p
      •  Cyc8p
      •  Gat3p
    • I then went to the link Generate Regulation Matrix on the yeastract database and copied and pasted the list of transcription factors above 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.

  1. 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).
    • I checked 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, it was deleted, making sure that I still had somewhere between 15 and 30 transcription factors in my 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.
  2. 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.

Summary of what you need to turn in for the individual Week 10 assignment

  1. Your individual journal page should have an electronic lab notebook recording your work. This includes the detailed methods specific to your analysis, your result files, the answers to any questions posed in the protocol above, a scientific conclusion, and the acknowledgments and references sections. Don't forget your paragraph which is a biological interpretation of your stem results.
  2. Upload your updated Excel spreadsheet to the wiki that has today's manipulations in it. Use the same filename as before so that the download link that you already (previous versions will still be available in the history).
  3. Append the screenshots of the stem results to the PowerPoint presentation that contains the p value table that you created for the Week 8 assignments. Each slide in the presentation should have a meaningful title that describes the main message of the slide.
  4. Zip together all of the tab-delimited text files that you created for and from stem and upload them to the wiki.
    • the file that was saved from your original spreadsheet that you used to run stem
    • each of the genelist and GOlist files for each of your significant profiles.
  5. Write a paragraph-length conclusion for this week's exercise.

Deliverables

File:Yeastract Matrix DB