Delta2D Report: Blotting
for Project 'Coverage Report'

Used approach: This is a 'Gel - Membrane / Western Blot' project.

The coverage between each Gel image and Western Blot image is computed with this formula:

Coverage (COV2) =	No. spots Western Blot Image	* 100%
	No. spots Total Protein Image + No. unmatched spots Western Blot Image

The coverage among the same kind of image (Gel, Western Blot) is computed with this formula:

Coverage (COV1) =	No. matched spots	* 100%
	No. matched spots + No. unmatched spots Image 1 + No. unmatched spots Image 2

Direct Warped Pairs (Warp Relations)

These image pairs have been warped to eliminate gel distortions.

Transfer Origin (Fused Image):	Fused Image using Max Intensity at 01_11_2016 11_35_38_845
Spot Detection Parameters
Local Background:	125
Average Spotsize:	41
Sensitivity:	20.0
# of originally detected spots:	916
# of manually added spots:	1
# of spots after spot editing:	917
# of canceled spots:	755
# of transferred spots:	162

Image pairs have been created in accordance with the 'Gel - Membrane / Western Blot' structure of this project.
Unmatched spots among filter settings in Delta2D.

Group columns are ordered in the same way as the group rows, i.e. the name of the image in column 2 of a certain group can be taken from the second row of the said group.

Reproducibility for Image Pairs in Groups

Group	N	Mean	RSD
Total Protein	0	VV N.A. %	VV N.A. %
2D WB	0	VV N.A. %	VV N.A. %

Statistics on Relative Coverage for Group Pairs

Total Protein Group	Western Blot Group	N	Mean	RSD
Total Protein	2D WB	1	41.98 %	0.00 %

Remarks

Analysis Procedure

1. The project may contain up to 9 groups (plus fused image group and internal standard group), further groups will be ignored.
2. Each group may contain up to 6 images, further images will be ignored.
3. Image names are not longer than 35 characters, gel numbers not longer than 2 characters.
4. The group containing fused images is named 'Fused Images'.
5. A group called 'Internal Standard' will be ignored as this group is assumed to contain just internal standard images.
6. Spot transfer to all images must be done from the same fused image.

Experiment Structure

Westernblots with multi fluorescence, maximal 3 Westernblot dyes. Total-Protein group must be the first group in the project.
Image pairs:

• each Total Protein Image with one Westernblot Dye N (N: 1...3)
  images in all groups must be sorted the same way to enable an appropriate assignment of each western blot image to the respective total protein image
• all possible pairs within each group
• Special coverage formula for image pairs Total Protein Image - Westernblot Dye
  Example:
  
  
  COV1 = Coverage formula for Gel - Gel approach
  

  Coverage (COV1) =	No. matched spots	* 100%
  	No. matched spots + No. unmatched spots Image 1 + No. unmatched spots Image 2

  COV2 = Coverage formula for Total Protein - Western Blot approach:

Coverage (COV2) =	No. spots Western Blot Image	* 100%
	No. spots Total Protein Image + No. unmatched spots Western Blot Image

• A number of 1 (k) group 'Total Protein' and 3 (l) groups with 'Westernblot Dyes' with 6 (m) images each results in 6*3+4*(6*6-6)/2=78 image pairs, i.e. the same number of report pages, general: m*l+(k+l)*(m*m-m)/2.

Report Content

1. Section 'Sample to Gel Assignment':
   • The section shows a table summarizing the sample to gel assignment used in this Blotting Report.
2. Section 'Experimental Setup':
   • The coverage formulas are automatically applied.
3. Section 'Fused Images from Group 'Fused Images'':
   • On one page a maximum of 6 fused images is described (configurable).
4. Section 'Transferred Spot Pattern':
   • The number of spots after spot editing is the sum of originally detected spots and manually added spots.
   • The number of transferred spots is the difference between spots after spot editing and canceled spots.
5. Section 'Fused Image 'Fused Image using Max Intensity at 01_11_2016 11_35_38_845'':
   • The image shows all spots and spot edit markers (if available).
   • Canceled spots are shown with dotted spot boundaries.
6. Section 'Image Pairs':
   • Includes data for the interesting image pairs for which the 'Coverage' table includes values.
   • Images show spot boundaries for interesting spots only, not for canceled spots.
   • Matched Spots are shown in blue, unmatched in green or red, respectively.
7. Section 'Coverage Table':
   • Coverage is calculated based on the formula as shown in the examples above.
   • Coverage table is split after a maximum of 2 leading columns and 14 value columns and/or 2 leading rows and 40 value rows (configurable).
   • Groups will not be split.
   • To reduce the width of coverage tables a legend can be introduced.
8. Section 'Results':
   • 'Reproducibility for Image Pairs in Groups' (1) and 'Statistics on Relative Coverage for Group Pairs' (2) are always based on the respective existing coverage values.
   • 'Mean' is the mean of coverage value of each image pair
   • 'RSD' (relative standard deviation) is the 'corrected sample standard deviation' (as defined in https://en.wikipedia.org/wiki/Standard_deviation), expressed in %.
   • 'N' is the number of image pairs (1) or the number of replicates (2)