ChIP-Seq Tutorial - Revision history

Julien: /* Score Promoters */

2014-04-21T21:15:12Z

‎Score Promoters

Julien: /* Export Result */

2014-04-21T21:15:03Z

‎Export Result

Julien: /* Extract Gene Promoters */

2014-04-21T21:14:46Z

‎Extract Gene Promoters

Julien: /* Isolate Peaks */

2014-04-21T21:14:27Z

‎Isolate Peaks

Julien: /* Filter Negative Values */

2014-04-21T21:14:07Z

‎Filter Negative Values

Julien: /* Compare Input to Input */

2014-04-21T21:13:48Z

‎Compare Input to Input

Julien: /* Compare Input to Input */

2014-04-21T21:13:24Z

‎Compare Input to Input

Julien: /* Normalize IP and Input */

2014-04-21T21:13:05Z

‎Normalize IP and Input

Julien: /* Load the Files */

2014-04-21T21:12:41Z

‎Load the Files

Julien at 22:25, 14 April 2014

2014-04-14T22:25:53Z

@@ Line 154: / Line 154: @@
 Then select ''Maximum Coverage'' method of calculation (figure 28). Note that the color of the promoters represents the scores associated to the promoters (as described in the gene layer section of the documentation). The result is shown in figure 29.
 [[image:ChIP-Seq_tutorial_score_exons3.png|center|frame|Figure 28: Method of Calculation]]
-[[image:ChIP-Seq_tutorial_score_exons4.png|center|frame|Figure 29: Score Exons Result]]
+[[image:ChIP-Seq_tutorial_score_exons4.png|center|thumb|800px|Figure 29: Score Exons Result]]
 <br style="clear: both" />

@@ Line 160: / Line 160: @@
 == Export Result ==
 The last thing we need to do is to export the result of our analysis. Right click on the track handler of the track with the gene layer. Select "Save As" in the gene layer submenu (figure 30). Choose where you want to save the track and make sure that the file type is set to Bed file.
-[[image:ChIP-Seq_tutorial_save_layer.png|center|frame|Figure 30: Save Layer]]
+[[image:ChIP-Seq_tutorial_save_layer.png|center|thumb|800px|Figure 30: Save Layer]]
 Once it's done, you can open the file that you created with a text editor such as notepad. You'll notice that the result file contains the position  (field 1 to 3) of the promoters, the name of the genes (field 4), the strand of the gene (field 6) as well as the scores of the promoters (field 5). For more details about the result file you can refer to the File Type section of the documentation.

@@ Line 133: / Line 133: @@
 When it's done, right click on the track handler of the gene track and select "Extract Intervals" in the RefSeq sub-menu (Figure 23).
-[[image:ChIP-Seq_tutorial_extract_promoters1.png|center|frame|Figure 23: Extract Intervals Menu]]
+[[image:ChIP-Seq_tutorial_extract_promoters1.png|center|thumb|800px|Figure 23: Extract Intervals Menu]]
 A dialog box will pop-up. We decide to define a promoter as a region that starts 500 bp before a gene start position and ends 500bp after. In order to do so, fill in the parameters as shown in figure 24.
@@ Line 139: / Line 139: @@
 You'll finally be asked to select the result track position in the track list. The result track represents only the promoters of the genes of the reference track (figure 25).
-[[image:ChIP-Seq_tutorial_extract_promoters3.png|center|frame|Figure 25: Gene Promoters (the track 4 contains only the gene promoters)]]
+[[image:ChIP-Seq_tutorial_extract_promoters3.png|center|thumb|800px|Figure 25: Gene Promoters (the track 4 contains only the gene promoters)]]
 <br style="clear: both" />

@@ Line 117: / Line 117: @@
 To do so, right click on the track handler, choose the "Input - Output" sub-menu and click on the "Find Peaks" option (figure 20).
-[[image:ChIP-Seq_tutorial_find_peaks1.png|center|frame|Figure 20: Find Peaks Operation]]
+[[image:ChIP-Seq_tutorial_find_peaks1.png|center|thumb|800px|Figure 20: Find Peaks Operation]]
 After the ''Find Peaks'' dialog opens, choose the "Island Finder" option on the left panel and set the parameters as shown on the figure 21.
@@ Line 125: / Line 125: @@
 You'll notice that the selected output is "Peak Summits". This means that for each island, the only selected window will be the one with greatest score on the island. This generally corresponds to the window with the highest probability of containing the binding site. Other software can be used to identify the exact sequence of the binding site. In addition to choosing the summit as the output, one could add the Island score or the  "start value". These two values are a much more quantitative estimate of the level of enrichment of the signal than the summit value.
 The result should be similar to what is shown on figure 22.
-[[image:ChIP-Seq_tutorial_find_peaks3.png|center|frame|Figure 22: Find Peaks Result (the track 4 contains only the summit of the peaks)]]
+[[image:ChIP-Seq_tutorial_find_peaks3.png|center|thumb|800px|Figure 22: Find Peaks Result (the track 4 contains only the summit of the peaks)]]
 <br style="clear: both" />

@@ Line 105: / Line 105: @@
 To filter the track you need to select the "Filter" option of the layer sub-menu (figure 17). On the left panel of the filter window select the threshold option. Set the parameters of the right panel as shown on figure 18. The figure 19 shows the result of the filter operation.
-[[image:ChIP-Seq_tutorial_filter1.png|center|frame|Figure 17: Filter Operation Menu]]
+[[image:ChIP-Seq_tutorial_filter1.png|center|thumb|800px|Figure 17: Filter Operation Menu]]
 [[image:ChIP-Seq_tutorial_filter2.png|center|frame|Figure 18: Filter Operation Window]]
-[[image:ChIP-Seq_tutorial_filter3.png|center|frame|Figure 19: Filter Operation Result (the negative values of the track 5 have been removed)]]
+[[image:ChIP-Seq_tutorial_filter3.png|center|thumb|800px|Figure 19: Filter Operation Result (the negative values of the track 5 have been removed)]]
 We now need to remove the background noise and to keep only the islands (the peaks).

@@ Line 93: / Line 93: @@
 [[image:ChIP-Seq_tutorial_subtract2.png|center|frame|Figure 13: Dialog to Choose the Second Track]]
 [[image:ChIP-Seq_tutorial_subtract3.png|center|frame|Figure 14: Dialog to Choose the Operation Type]]
-[[image:ChIP-Seq_tutorial_subtract4.png|center|frame|Figure 15: Result of the Subtraction]]
+[[image:ChIP-Seq_tutorial_subtract4.png|center|thumb|800px|Figure 15: Result of the Subtraction]]
 We can now change the display properties of the track by double clicking on the track handler of the layer resulting from the subtraction like we did for the two previous tracks. From the same window we can also change the layer name as shown in figure 16.
-[[image:ChIP-Seq_tutorial_layer_name.png|center|frame|Figure 16: Change Layer Name]]
+[[image:ChIP-Seq_tutorial_layer_name.png|center|thumb|800px|Figure 16: Change Layer Name]]
 <br style="clear: both" />

@@ Line 90: / Line 90: @@
 We choose to subtract rather than divide (as would be done in a PCR experiment) because in chip-Seq experiments the input sample is not enriched wile the IP might be enriched 10 to 20 time or more. As a result, to have comparable coverage the input would have to be sequenced 10 to 20 times deeper than the IP sample (which would be very expensive). This lower coverage for the input control causes a sampling error that can drastically affect the results if the data are normalised by calculating the ratio IP/IN.  The sampling error problem on the input  increase when the binning size decrease (when the window size used loading the track decrease). An alternative to subtracting the input from the IP track is to smooth the input track in order to attenuate the sampling error on that track. That can be done in GenPlay using either the moving average or the Gaussian smoothing operation. In practice to decrease the sampling error, we find that smoothing  over 5 to 10 kb is necessary. Finally, whatever the method use, some of the peak present in the IP track might not have been completely eliminated from the IP track, or some artifact might be present only in the IP track. If that is the case, one can also eliminate the outliers by simply using the filter to eliminate the highest peak. This can be done easily in GenPlay using either either absolute or relative values in the filter operation. We find that there are no optimal method to analyse all tracks and that it is best for the users to proceed by trial and error and exercise their judgment in performing these operations.
-[[image:ChIP-Seq_tutorial_subtract1.png|center|frame|Figure 12: Two Tracks Operation Menu]]
+[[image:ChIP-Seq_tutorial_subtract1.png|center|thumb|800px|Figure 12: Two Tracks Operation Menu]]
 [[image:ChIP-Seq_tutorial_subtract2.png|center|frame|Figure 13: Dialog to Choose the Second Track]]
 [[image:ChIP-Seq_tutorial_subtract3.png|center|frame|Figure 14: Dialog to Choose the Operation Type]]

@@ Line 61: / Line 61: @@
 Once it's done repeat the operation with the input track.
-[[image:ChIP-Seq_tutorial_normalize1.png|center|frame|Figure 8: Normalize Menu]]
+[[image:ChIP-Seq_tutorial_normalize1.png|center|thumb|800px|Figure 8: Normalize Menu]]
 [[image:ChIP-Seq_tutorial_normalize2.png|center|frame|Figure 9: Normalize Dialog Box]]

@@ Line 19: / Line 19: @@
 We now need to load the IP data. To achieve that you will need to right click on the track handler of the 1st row. A popup menu should appear, select the ''Add Layer(s)'' option (Figure 3).
-[[image:ChIP-Seq_tutorial_add_layer.png|center|frame|200px|Figure 3: Add Layer(s) Menu]]
+[[image:ChIP-Seq_tutorial_add_layer.png|center|thumb|800px|Figure 3: Add Layer(s) Menu]]
 Then select the IP file that you downloaded earlier. You will be prompted to choose a layer type. Choose ''Variable or Fixed Window Layer''.
@@ Line 32: / Line 32: @@
 Click on Okay and wait for the track to load. That can take more than a minute.
 Once the data is loaded, double click on the handle of the track 1 to adjust the display options. Check ''Show horizontal lines', uncheck ''Auto rescale'' and enter 100 for the ''Maximum Score'' (figure 5).
-[[image:ChIP-Seq_tutorial_track_options.png|center|frame|Figure 5: Track Options]]
+[[image:ChIP-Seq_tutorial_track_options.png|center|thumb|800px|Figure 5: Track Options]]
 You can now look at your data by zooming using the mouse wheel and moving within the chromosome by pressing the left button of the mouse on the track and releasing it after moving the mouse on the left or on the right.
-[[image:ChIP-Seq_tutorial_IP_layer_result.png|center|frame|Figure 6: IP Layer Loaded]]
+[[image:ChIP-Seq_tutorial_IP_layer_result.png|center|thumb|800px|Figure 6: IP Layer Loaded]]
 <br style="clear: both" />

@@ Line 9: / Line 9: @@
 You can enter "ChIP-Seq Tutorial" for the name, select the mammal clade and human genome (figure 1).
 [[image: ChIP-Seq_tutorial_project_frame.png|center|frame|Figure 1: New Project Window]]
-Then, click on the tool box button on the assembly line. A new window will appear allowing you to select chromosomes. For this tutorial we will work only on the first chromosome so loading times are shorter and the amount of memory needed is smaller. You can select all the other chromosome and then click on the unselect button (figure 2).
+Then, click on the tool box button on the assembly line. A new window will appear allowing you to select chromosomes. For this tutorial we will work only on the first chromosome so loading times are shorter and the amount of memory needed is smaller. You can select all the other chromosomes and then click on the unselect button (figure 2).
 [[image: ChIP-Seq_tutorial_project_chromosomes.png|center|frame|Figure 2: Project Chromosomes]]