Dahlquist:GenePix Pro Software Protocol

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(link to new page for next steps in analysis)
Current revision (14:51, 14 May 2015) (view source)
(Using GenePix Pro: clarified version of software manual)
 
(20 intermediate revisions not shown.)
Line 1: Line 1:
== Using GenePix Pro ==
== Using GenePix Pro ==
-
=== Biology 478/Spring 2013 students: ===
+
[http://www.openwetware.org/images/8/88/GenePix.pdf GenePix Pro 6.0 Software Manual]
-
* 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]
+
=== Scanner Settings ===
-
* Download both the 532 and 635 wavelength files for your group onto your Desktop.
+
 
-
* Download the file "Ontario_Y6.4Kv7.GAL" to your Desktop.
+
[[Image:Dahlquist_GenePixPro_versioninfo_20150506.jpg]]
-
** '''''HINT: Right-click on the link and select the menu item "Save target as" or "Save link as"''''' to download the file. If you just click on the link, your browser will attempt to open it without downloading
+
* 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 ===
=== Gridding and Generating Intensity Data ===
-
* Launch GenPix Pro (select Analysis Only)
+
* 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
-
** Navigate to the folder containing your .tif images (should be the Desktop)
+
** 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
Line 21: Line 44:
* 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
-
** Select the file "Ontario_Y6.4Kv7.GAL" (should be on the Desktop)
+
** Select the file [[Media:Ontario_Y6.4Kv7.GAL | 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
** 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
-
** Click on the icon that looks like a compass, or a circle with cross-hairs, and select Find Array
+
** Click on the icon that looks like a compass, or a circle with cross-hairs, and select Find Array, Find All blocks, Align Features
-
** Click on the icon again, and select Find All Blocks
+
*** Alternately, you can do this in three steps by:
-
** Click on the icon again, and select Align Features in All Blocks
+
**** 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)
* 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)
-
* In the Results tab, click on the button to Configure Normalization
+
* 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.
-
** Make sure that "Ratio-based" is selected and it says that the "Ratio of medians" of "all of the features" is equal to "1"
+
-
** Click the Normalize button
+
-
* Click on the File icon on the right hand side and select, Save Results As, and save your results.
+
=== Generating an Array Quality Report ===
=== Generating an Array Quality Report ===
   
   
-
* Launch GenePix Pro (select Analysis Only)
+
* 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
Line 59: Line 82:
* Select Print
* Select Print
* Save on the Desktop
* Save on the Desktop
-
* Name the file: Chip #_Year-Month-Date
+
* 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
 +
<!--
== Looking at the raw data ==
== Looking at the raw data ==
Line 71: Line 95:
* 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

Personal tools