Protocol Written by Dr. Terrie Rife and Jeff Kucharski
Revised April 2003 by JMU Students-- Scott Dart, Fatima Diab, Karah Glascock, and Sarah Javaid
James Madison University
Link to the OnLine GeneSpring Tutorial
This protocol is divided into the following sections-
1. Scanalyze to Excel
2Excel to GeneSpring
3. Things to do in GeneSpring
4. Rearranging the
yeast data to make the genelist match the Scanalyze Spread
Scanalyze to Excel:
1. In Scanalyze set up and grid all of your data (See the Scanalyze Protocol).
2. Once data is gridded press save data to generate an Excel spread sheet.
3. Go to http://www.bio.davidson.edu/Biology/GCAT/GCATprotocols.html. Click on Yeast resources and then download the gene file for your ISB chip (know the number marked on your chip and click on the range it falls within).
4. Open Excel spreadsheet and add in gene names and flags from your datafile provided by Dr. Campbell (the datafile was downloaded in Step 3). If you plan on clustering your data using GeneSpring it is best for these columns to all be put in the same position. So please add the column "ORF name" in to the end of your Excel file first and then add in the column for the "Flag" that tell how well your slide was printed. Make sure the right gene name and flag matches with the data in scanalyze. You may have to rearrange your data from Dr. Campbell depending on how the slides are oriented (see section 4 below). To get the data for ORF name, go to the gene file that you downloaded in Step 3 under yeast resources. There should be an ORF name in that file (if not, the ORF name usually contains data like the following: YBR124W or YBR121C). To get the data for Flag, go to the gene file that you downloaded in Step 3 under yeast resources. There should be a Flag in the file (if not, ignore). You may also want to add another column after the Flag (if its there) called Gene Name to be able to locate the common names of genes. To get the data for Gene Name, go to the gene file that you downloaded in Step 3 under yeast resources. There should be a Gene Name column in that file (if not, the Gene Name column contains data like the following: ZMS1 or ZWF1). Close the gene file that you downloaded in Step 3 since it is no longer needed.
5. Next you need to
do some reorganization of your file so you can see it better when you
try to load your file into GeneSpring. All of the column headings
that are at the top of the Scanalyze data need to be moved down. Here
is a little bit of the graph to give you an idea what it should look
like after it is moved down. We have highlighted in red the line of
column headings that need to be brought down from above the remarks
to the top of the data. Remark Date 11-29-01 Remark Header Spot Grid Top Left Spot 1 1 149 5295
6. One final step in reorganization must also be done if you intend to cluster data from this slides with different color scheme. You must arrange the flourescent signals and background read by Scanalyze from both sides of the chip so that they are in the same position. The green intensity can be found in a column labeled CH1I and the green background is found in the column called CH1B. The red intensity is found in the column CH2I and the red background is found in the column CH2B.
Ie. If you performed some of your experiments labeling for example WT with green and some where you labeled WT with red you will need to move these columns so they are both in the same position. Everyone should have the WT intensity column first whether it is red or green then the WT background then the Mutant intensity column then the mutant background. For example, if your WT was labeled red, then the column CH2I (red intensity) and CH2B (red background) would be moved ahead of CH1I (green intensity) and CH1B (green background) if needed. If your WT was green, then the column CH1I (green intensity) and CH1B (green background) would be moved ahead of CH2I (red intensity) and CH2B (red background) if needed.
7. Finally, you will need to save your new revised Excel file as a tab-delimited file in order to open it in GeneSpring. The extension of the file name is .DAT.
1. Open GeneSpring and login under guest. Go under FILE to IMPORT DATA. Choose the file you just updated and revised (it is the tab-delimited file with the .DAT extension). Select the Yeast genome to work with and press next. It might take a few minutes to extract the file and a window appears labeled "Column Editor".
2. Now you will need to spend some time telling GeneSpring about your data. You will notice that right above your column headings there is a new column marked "Click to Set". This is the column you use to tell the computer about your data. If you click on the Click to Set you will notice you will pull down a whole menu of options to select. There are five different columns that you need to change the "Click to Set" remark in for GeneSpring to be able to analyze your data.
Here are the ones you must change
ORF NAME -- you will need to tell the computer this is the gene name by changing the unassigned above it to "gene identifier". There is already a column heading Gene Name in this menu: DO NOT CHANGE THAT LABEL. Leave it as unassigned.
FLAGS -- you will need to tell the computer this is the flag by putting the word "Flag" in the unassigned spot above it. If there is no data for flag, don't put flag in the unassigned spot. In other words, if you did not flag any spots in ScanAlyze OR there was no FLAG column in your downloaded gene file (Step 3), then ignore this step.
CH1I -- CH1I is the green intensity. You will need to let the computer know if this is your "Control Chnl" (WT) or "Signal" (mutant signal). If you labeled your WT as green, then you will need to change the "Click to Set" to "Control Chnl" in the pull-down menu. If you labeled your mutant as green, then you will need to change the "Click to Set" to "Signal".
CH1B -- CH1B is the green background. you will need to let the computer know if this is your "Control Background" (WT) or "Signal Background" (mutant). If you labeled your WT as green, then you will need to change the "Click to Set" to "Ctrl Chnl Bkgd" in the pull-down menu. If you labeled your mutant as green, then you will need to change the "Click to Set" to "Signal Background".
You will then need to do the same things for CH2I (red intensity) and CH2B (red background). For example, if your WT was labeled green, then your CH1I and CH1B would be changed from "Click to Set" to "Ctrl Chnl" and "Ctnl Chnl Bkgd" respectively. Now, for this example, you will need to go to CH2I and change the "Click to Set" to "Signal" in the pull-down menu. Also, change CH2B from "Click to Set" to "Signal Background". Now if your WT was labeled red, then your CH1I and CH1B would be changed from "Click to Set" to "Signal" and "Signal Background" and your CH2I and CH2B would be changed to "Ctrl Chnl" and "Ctrl Chnl Bkgd" respectively.
3. If you don't have the flags, don't worry about this step and skip to the next step. You will also want to tell GeneSpring how to read your flags if you have them. At the bottom right corner, is a box that allows the flags to entered. In the Flag Values box at the bottom right corner, enter a P for Pass Flag, an A for Absent, and M for Marginal. Now GeneSpring will be able to understand how well your spots were printed.
4. Click the Next button. In the next window GeneSpring will allow you to load any other files so if you have several files that you want to cluster, now is the time to load them in. You will have to name all of your files but now all of them are loaded into GeneSpring under the Experiments folder and even if you turn the computer on and off you should never have to do these steps again. The data downloaded will only be on the computer where all these steps were done.
5. GeneSpring will now ask you if you would like to create a new experiment, click yes. Now type in a name for your experiment and click save. GeneSpring will now ask you to define normalizations, parameters, and the default interpretation, just click close and now your data should be in view.
1. Under Tools, click Filter genes.
2. Click on experiments folder-Find your experiment
3. Click on + , Click on + of Default Interpretation
4. Right click on the file name, choose expression restriction.
5. Enter the minimum
and maximum you want.
Rearranging the Yeast data to make the GeneList match the Scanalyze Spread Sheet - if you choose not to rotate the .Tiff images before you put them in Scanalyze - make sure you do the following.
1. In Scanalyze make
sure your grids are ordered as follows. Grids numbered 45-48 are the
ones that are cut off. They should still be the same size but you
will have to move them so that part of the grid is not in the picture
since Dr. Campbell can't read all of the data.
45 41 37 33 29 25 21 17 13 9 5 1 46 42 38 34 30 26 22 18 14 10 6 2 47 43 39 35 31 27 23 19 15 11 7 3 48 44 40 36 32 28 24 20 16 12 8 4
2. Now with Gene list give to us by Dr. Campbell. Highlight all of the genes in metarow 1. Hit the sort key in Excel. It is under the data function.
Now we will do three sorts simultaneously. For the first sort sort by metacolumn in ascending order. Then sort by column in ascending order and thirdly you will want to sort by row in descending order.
You will need to do this 12 times for each of the meta rows.
Because you highlighted the row and will first sort by metacolumn you will first have- grid 1 then grid 2 then grid 3 then grid 4 data as all these are in meta row 1 of the original data. The second two simultaneous sorts will cause you to read from right to let in grid 1 what used to be column 1 then grid 1 what used to be column 2...