Dahlquist:GenePix Pro Software Protocol

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== Using GenePix Pro ==
== Using GenePix Pro ==
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=== Biology 478/Spring 2013 students: ===
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[http://www.openwetware.org/images/8/88/GenePix.pdf GenePix Pro 6.0 Software Manual]
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* Follow this link to access your microarray image and .gal files from LionShare:  [https://lionshare.lmu.edu/Users/kdahlqui/BIOL478 https://lionshare.lmu.edu/Users/kdahlqui/BIOL478]
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=== Scanner Settings ===
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* [https://lionshare.lmu.edu/xythoswfs/webview/fileManager.action?entryName=/Users/kdahlqui/BIOL478&stk=EDEAEDD3B58C2E2&msgStatus=8%20documents%20have%20been%20successfully%20uploaded%20to%20the%20folder%2C%20BIOL478.]
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* Download both the 532 and 635 wavelength files for your group onto your Desktop.
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[[Image:Dahlquist_GenePixPro_versioninfo_20150506.jpg]]
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** '''''HINT: Right-click on the link and select the menu item "Save to Desktop"''''' to download the file.
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* Version information for the GenePix Pro software used to scan chips in the Dahlquist Lab.
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* Download the file "Ontario_Y6.4Kv7.GAL" to your Desktop.
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[[Image:Dahlquist_GenePixPro_HardwareSettings_20150506.jpg]]
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* Hardware settings:
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** We use a pixel size of 10 microns.
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** We use two lines to average.
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** We use a focus position of 0.
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** We use Auto PMT settings at 100% power (see below).
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[[Image:Dahlquist_GenePixPro_AutoPMTSettings_20150506.jpg]]
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* Auto PMT settings:
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** Saturation tolerance is set to the default value of 0.05%.
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[[Image:Dahlquist_GenePixPro_ScanSettings_20150506.jpg]]
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* Alignment Options:
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** Find irregular features
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** Resize features during alignment to minimum diameter 33% and maximum diameter 200%
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** Flag features that fail background threshold criteria as Not Found
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** Estimate warping and rotation when finding blocks
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** Automatic Image Registration: Max translation 10 pixels.
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* Saving scanned images:
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** Create a folder on the C: drive called "MicroarrayData_yyyymmdd" where yyyymmdd is the current date.
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* Save images to this folder as "Single-image TIFF Files (*.tif)".  Do  '''not''' use TIFF LZW compression (lossless).
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** Name files with a Date prefix and Barcode prefix.
=== Gridding and Generating Intensity Data ===
=== Gridding and Generating Intensity Data ===
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* Launch GenPix Pro (select Analysis Only)
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* Launch GenPix Pro 6.1 (select Analysis Only)
* On the right hand side of the screen, select the File icon
* On the right hand side of the screen, select the File icon
** Select File > Open Images
** Select File > Open Images
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** Navigate to the folder containing your .tif images (should be the Desktop)
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** Navigate to the folder containing your .tif images
** Hold down the Control key while clicking to select both the 532 and 635 wavelength files
** Hold down the Control key while clicking to select both the 532 and 635 wavelength files
** Click on the Open button
** Click on the Open button
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* On the right hand side of the screen, select the File icon
* On the right hand side of the screen, select the File icon
** Select Load Array List
** Select Load Array List
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** Select the file "Ontario_Y6.4Kv7.GAL" (should be on the Desktop)
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** Select the file [[Media:Ontario_Y6.4Kv7.GAL | Ontario_Y6.4Kv7.GAL]]
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*** To download this file, right-click on the link and select "Save link as..." from the context menu that appears.
** Click the Open button
** Click the Open button
* Using the icon that has an arrow and a square, drag the grids so that they are approximately aligned with the top block of spots
* Using the icon that has an arrow and a square, drag the grids so that they are approximately aligned with the top block of spots
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** Click on the icon that looks like a compass, or a circle with cross-hairs, and select Find Array
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** Click on the icon that looks like a compass, or a circle with cross-hairs, and select Find Array, Find All blocks, Align Features
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** Click on the icon again, and select Find All Blocks
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*** Alternately, you can do this in three steps by:
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** Click on the icon again, and select Align Features in All Blocks
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**** Click on the icon that looks like a compass, or a circle with cross-hairs, and select Find Array
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**** Click on the icon again, and select Find All Blocks
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**** Click on the icon again, and select Align Features in All Blocks
* On the right hand side of the screen, select the icon that looks like a table of data (it says B, C, R at the top of it)
* On the right hand side of the screen, select the icon that looks like a table of data (it says B, C, R at the top of it)
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* In the Results tab, click on the button to Configure Normalization
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* Click on the File icon on the right hand side and select, Save Results As, and save your results as type "GenePix Pro Files (*.gpr)".  Save a JPEG image containing all analyzed features.
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** Make sure that "Ratio-based" is selected and it says that the "Ratio of medians" of "all of the features" is equal to "1"
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** Click the Normalize button
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* Click on the File icon on the right hand side and select, Save Results As, and save your results.
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=== Generating an Array Quality Report ===
=== Generating an Array Quality Report ===
   
   
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* Launch GenePix Pro (select Analysis Only)
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* Launch GenePix Pro 6.1 (select Analysis Only)
* On the right hand side of the screen, select the File icon
* On the right hand side of the screen, select the File icon
* File > Open Results
* File > Open Results
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* Select Print
* Select Print
* Save on the Desktop
* Save on the Desktop
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* Name the file: Chip #_Year-Month-Date
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* Name the file: ChipBarcode#_yyyymmdd
* The PDF file will either open on its own or you need to open it from the file you saved it to
* The PDF file will either open on its own or you need to open it from the file you saved it to
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<!--
== Looking at the raw data ==
== Looking at the raw data ==
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* Repeat with ''SWI4'' ([http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=SWI4 ''SWI4'' page at the ''Saccharomyces'' Gene Database])
* Repeat with ''SWI4'' ([http://www.yeastgenome.org/cgi-bin/locus.fpl?locus=SWI4 ''SWI4'' page at the ''Saccharomyces'' Gene Database])
* We are expecting that ''NSR1'' should be expressed and that ''SWI4'' should not because it has been deleted from the yeast genome.  Is this the case?
* We are expecting that ''NSR1'' should be expressed and that ''SWI4'' should not because it has been deleted from the yeast genome.  Is this the case?
 +
-->
== Next Steps ==
== Next Steps ==
* Go to the [[BIOL478/S13:Microarray Data Analysis | Microarray Data Analysis]] page for the next steps.
* Go to the [[BIOL478/S13:Microarray Data Analysis | Microarray Data Analysis]] page for the next steps.

Current revision

Contents

Using GenePix Pro

GenePix Pro 6.0 Software Manual

Scanner Settings

Image:Dahlquist_GenePixPro_versioninfo_20150506.jpg

  • Version information for the GenePix Pro software used to scan chips in the Dahlquist Lab.

Image:Dahlquist_GenePixPro_HardwareSettings_20150506.jpg

  • Hardware settings:
    • We use a pixel size of 10 microns.
    • We use two lines to average.
    • We use a focus position of 0.
    • We use Auto PMT settings at 100% power (see below).

Image:Dahlquist_GenePixPro_AutoPMTSettings_20150506.jpg

  • Auto PMT settings:
    • Saturation tolerance is set to the default value of 0.05%.

Image:Dahlquist_GenePixPro_ScanSettings_20150506.jpg

  • Alignment Options:
    • Find irregular features
    • Resize features during alignment to minimum diameter 33% and maximum diameter 200%
    • Flag features that fail background threshold criteria as Not Found
    • Estimate warping and rotation when finding blocks
    • Automatic Image Registration: Max translation 10 pixels.
  • Saving scanned images:
    • Create a folder on the C: drive called "MicroarrayData_yyyymmdd" where yyyymmdd is the current date.
  • Save images to this folder as "Single-image TIFF Files (*.tif)". Do not use TIFF LZW compression (lossless).
    • Name files with a Date prefix and Barcode prefix.

Gridding and Generating Intensity Data

  • Launch GenPix Pro 6.1 (select Analysis Only)
  • On the right hand side of the screen, select the File icon
    • Select File > Open Images
    • Navigate to the folder containing your .tif images
    • Hold down the Control key while clicking to select both the 532 and 635 wavelength files
    • Click on the Open button
  • Use the brightness and contrast sliders on the upper left hand side to adjust your image to see all of the spots
  • You can use the magnifying glass icon to zoom in on an area of the image, and the magnifying glass return icon to go back to the whole chip image
    • Zoom in so that you can see the entire top block of grids on your screen
  • On the right hand side of the screen, select the File icon
    • Select Load Array List
    • Select the file Ontario_Y6.4Kv7.GAL
      • To download this file, right-click on the link and select "Save link as..." from the context menu that appears.
    • Click the Open button
  • Using the icon that has an arrow and a square, drag the grids so that they are approximately aligned with the top block of spots
    • Click on the icon that looks like a compass, or a circle with cross-hairs, and select Find Array, Find All blocks, Align Features
      • Alternately, you can do this in three steps by:
        • Click on the icon that looks like a compass, or a circle with cross-hairs, and select Find Array
        • Click on the icon again, and select Find All Blocks
        • Click on the icon again, and select Align Features in All Blocks
  • On the right hand side of the screen, select the icon that looks like a table of data (it says B, C, R at the top of it)
  • Click on the File icon on the right hand side and select, Save Results As, and save your results as type "GenePix Pro Files (*.gpr)". Save a JPEG image containing all analyzed features.

Generating an Array Quality Report

  • Launch GenePix Pro 6.1 (select Analysis Only)
  • On the right hand side of the screen, select the File icon
  • File > Open Results
  • Select the desired file
  • Select Report tab at the top of the screen
  • On the left side of the screen, under Navigate, select the back arrow or home icon
  • Select Array Quality Control
  • Adjust Vital Statistic Thresholds values to:
Quality Control Limits
Median signal to background > 2.5
Mean of Median background < 500
Median Signal to Noise	> 4
Median % >B + 1 StdDev	> 90
Feature variation < 0.5
Background Variation < 1.2
Features with Saturated pixels	< 3.3%
Not Found < 18%
Bad Features < 7%
  • Select Start
  • Select Show Printable Version
  • File > Print
  • Under Select Printer > Select Adobe PDF
  • Select Print
  • Save on the Desktop
  • Name the file: ChipBarcode#_yyyymmdd
  • The PDF file will either open on its own or you need to open it from the file you saved it to


Next Steps

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