Analyzing and Visualizing Your Gene Regulatory Networks: closed paren
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Revision as of 06:19, 9 April 2015
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** Link to this assignment from your journal entry.
** Link to this assignment from your journal entry.
** Don't forget to add the "BIOL398-04/S15" category to the end of your wiki page.
** Don't forget to add the "BIOL398-04/S15" category to the end of your wiki page.
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+
For your assignment this week, you will keep an '''''electronic laboratory notebook''''' on your individual wiki page that records all the manipulations you perform on the data and the '''''answers to the questions''''' throughout the protocol. We will be working on the protocols in class on Thursday, April 9. Whatever you do not finish in class will be homework to be completed by the Week 12 journal deadline.
=== Using YEASTRACT to Infer which Transcription Factors Regulate a Cluster of Genes ===
=== Using YEASTRACT to Infer which Transcription Factors Regulate a Cluster of Genes ===
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# Answer the following questions:
# 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"?'''''
#* 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"?'''''
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#* '''''List the "significant" transcription factors on your wiki page, along with the % in user set, % in YEASTRACT, and p value.'''''
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#* '''''List the "significant" transcription factors on your wiki page, along with the
corresponding "
% in user set
"
,
"
% in YEASTRACT
"
, and
"
p value
"
.'''''
-
#* '''''Are
Cin5
,
Gln3
,
Hmo1
, and
Zap1
on the list?'''''
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#
*
* '''''Are
CIN5
,
GLN3
,
HMO1
, and
ZAP1
on the list?'''''
-
# For the mathematical model that we will build in class, 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 will
use
the "significant" transcription factors
from
your
analysis above
and add CIN5, GLN3, HMO1, and ZAP1 if they are not in your list.
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# For the mathematical model that we will build in class, 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.
-
* Go back to the YEASTRACT database and follow the link to ''[http://www.yeastract.com/formgenerateregulationmatrix.php Generate Regulation Matrix]''.
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#*
You
and your partner
will
need to analyze
the
same gene regulatory network for your modeling project. Compare the lists of
"significant"
factors that you and your partner generated. '''''How many of the
transcription factors
appear in both of
your
lists?''''' You will use these transcription factors
and add CIN5, GLN3, HMO1, and ZAP1 if they are not in your list
. Use your discretion to add transcription factors until you reach a list of 15-30 factors. 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
.
-
*
* Copy and paste the list
above plus the additional
transcription factors you identified into both the "Transcription
Factor
" field and the "ORF/Genes" field.
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#
* Go back to the YEASTRACT database and follow the link to ''[http://www.yeastract.com/formgenerateregulationmatrix.php Generate Regulation Matrix]''.
-
*
* Uncheck the box for
"
Indirect Evidence
"
and select "JPEG" from
the
drop-down menu for
the "
Output Image
"
.
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#
* Copy and paste the list
of
transcription factors you identified
(plus CIN5, GLN3, HMO1, and ZAP1)
into both the "Transcription
factors
" field and the "
Target
ORF/Genes" field.
-
** Click the "Generate" button.
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#
*
We are going to generate several regulation matrices, with different
"
Regulations Filter
"
options.
-
* In the results window that appears, click on the link to the "
RegulationMatrix"
file that appears and save it to your Desktop
.
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#** For
the
first one, accept
the
defaults:
"
Documented", "DNA binding '''plus''' expression evidence
"
-
* Click on the "Image" link to see the diagram of the network
.
Save the image
file
(
you can
copy and paste
it
to your PowerPoint file or upload it to
the
wiki)
.
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#
** Click the "Generate" button.
-
*
We will use the matrix file as an input
to
your model next week
.
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#*
* 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
.
-
**
You will need
to
check it to make sure that
the
new transcription factors
you
added
to the
network actually have connections
to the
network
.
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#
*
* Repeat these steps
to
generate a second regulation matrix, this time applying the Regulations Filter "Documented", "'''Only''' DNA binding evidence"
.
-
*
*
Open the file in Excel. It will not open properly in Excel because a semicolon was used as the column
delimeter
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.
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#
**
Repeat these steps a third time
to
generate a third regulation matrix, this time applying
the
Regulations Filter "Documented", DNA binding '''and''' expression evidence".
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*
* Save this file in Microsoft Excel format (
.xls or
.xlsx).
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-
*
* Check to see that the
new
transcription factors
that you added
are connected to at least one of the other factors by making sure that there is at least one "1" in a row or column for that transcription factor. If
the new
factor
you added
is not connected, delete its row and column from the matrix. Make sure that you have
added at least three new
factors
to the
network
. If you haven't, then you will need to choose other new transcription factors until you do
.
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=== Analyzing and Visualizing Your Gene Regulatory Networks ===
-
*
* To work with our model
, we need to transpose the matrix. Insert a new worksheet into your Excel file. Go back 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.
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*
Make sure
that your
wiki assignment page includes
your PowerPoint
file
to
which
you
saved your screenshots
and
your
"
RegulationMatrix
" file.
+
We will analyze the regulatory matrix files
you
generated above in Microsoft Excel
to
determine which one will be appropriate to pursue further in
the
modeling.
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# 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.
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#
*
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.
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#
* Save this file in Microsoft Excel
workbook
format (.xlsx).
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#
* 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
.
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#
*
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.
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#
*
In cell A1, copy and paste the text "rows genes affected/cols genes controlling".
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# Now we will look at some of the network properties. Again, repeat these steps for each of the three gene regulatory matrices you generated above.
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#* Create a new worksheet and call it "degree". Copy and paste your adjacency matrix from the "network" sheet into this new worksheet.
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#* In the first empty cell in column A, type "Out-degree". In the cell to the right of
that
in Column B, type the equation <code>=SUM(</code> and select the range of cells in column B that has 1's and 0's in it, close the parentheses, and press Enter. This quantity is the number of genes that the transcription factor in that column is controlling, or the out-degree. Copy and paste that equation across all of the columns.
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#* In Cell 1 of the first empty column to the right of the adjacency matrix, type "In-degree". In Cell 2 of this column, type the equation <code>=SUM(</code> and select the entire row of 1's and 0's, close the parentheses, and press Enter. This quantity is the number of transcription factors that regulate the gene in that row, or the in-degree. Copy and paste the equation down the entire column, including the row that contains the out-degree sums.
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#* The number in the lower right-hand corner, the sum of sums, is the total number of edges in the adjacency matrix. We would like to see about 50 (40-60 or so) edges in the matrix. If the matrix is too dense, it will slow down the modeling program and be difficult to estimate the parameters in the model.
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#* We want to plot the degree distributions for each of
your
gene regulatory networks. In the "degree" worksheet, create three columns to the right called "Frequency", "In-degree total", and "Out-degree total". In the "Frequency" column, number sequentially from 1 to the largest degree number in your calculations above. In the "In-degree total" column, type the number of genes with that in-degree for each of the frequencies. In the "Out-degree total" column, type the number of genes with that out-degree for each of the frequencies.
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#* Select the "Frequency", "In-degree total", and "Out-degree total" columns. Go to the "Insert" tab and select the column chart type to insert a plot of the degree distribution. Copy and paste the charts for each gene regulatory matrix into
your PowerPoint
presentation.
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# Now we will visualize what these gene regulatory networks look like with the GRNsight software.
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#* Go
to
the [http://dondi.github.io/GRNsight/ GRNsight] home page (
you
can either use the version on the home page or the [http://dondi.github.io/GRNsight/beta.html beta version], which has slightly different visualization properties).
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#* Select the menu item File > Open
and
select one of 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. Repeat with the other two regulation matrix files. You will want to arrange the genes in the same order for each screenshot so that the matrices can be easily compared.
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# Write a paragraph discussing the results of today's work. In particular, which of the three regulation matrices does your group want to use for the modeling project and why?
== Shared Journal Assignment ==
== Shared Journal Assignment ==
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* Add the "BIOL398-04/S15" category to the end of the wiki page (if someone has not already done so).
* Add the "BIOL398-04/S15" category to the end of the wiki page (if someone has not already done so).
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=== Reflection ===
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# What aspect of this assignment came most easily to you?
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# What aspect of this assignment was the most challenging for you?
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# What (yet) do you not understand?
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