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SpotMap – Technical Specification

Technical Specification

SpotMap addresses the challenges of analysing 2D gels and Western blots. Images are quality checked on upload to address any potential challenges with the analysis. Positional variation in the spot patterns of the 2D gel and blot can make identification of corresponding spots challenging, alignment helps to overcome this. A single spot pattern is created to easily identify the corresponding spots on each image and results are clearly presented for easy reporting.

PC Specification

Windows Vista, 7, 8 and 10.
Recommended minimum: 4 GB RAM.
Performance is dependent on image file sizes.

SpotMap supports multi-core CPUs and 64-bit versions of Windows which are recommended for maximum performance.

Welcome to SpotMap, your essential partner for HCP coverage analysis via 2DE

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Image Formats

We recommend using the following image file formats:

  • .tiff
  • .gel
  • .mel

SpotMap can also analyse data from these file formats:

  • .png
  • .jpg

Analysis

Alignment takes two images and overlays them in the same coordinate space so that a spot on the image to be aligned will be in the same location as the matching spot on the target image. It makes it possible to accurately compare images by removing any positional variation introduced during the gel running, Western blotting and imaging processes.

What features are available in the align mode to aid alignment?

  • Alignment can be completed automatically.
  • Manual vectors are added by simple click, drag and drop.
  • Individual or multiple vectors can be easily deleted.
  • The alignment vectors added to one image can be copied to another, this feature is particularly useful when used with DIGE techniques.

How do I align my images in SpotMap? 

How do I check my Alignment is correct? 

How do I know that I have completed Alignment?

Spot Detection

A master spot map identifying the location of all spots on the gel and blot are created through spot detection. Spots on each image are categorized as present or absent from this map. The present or absent settings are used to identify spots common, missing or additional to the images in a comparison and measure % coverage.

Why is a single spot map used for the analysis?

Using a single spot map allows you to directly compare matching spots/features between images and easily identify spots absent from that given image. Alignment places corresponding spots in the same coordinate space on the images, this allows a single spot map to be used.

How is the single spot map generated?

User should detect spots on the gel first as stained gels are often more defined then a western blot image. The spot outlines are then transferred to the blot (this should be done after alignment as it helps to see where the spots exist on the blot) then you can detect spots on the blot. This will give you one single spot map.

Watch our video to see how SpotMap creates a single spot map.

Why are some spot edits required?

Sometimes the automatic spot detection results need to be refined. Spot outlines around the edge can be removed. Spot outlines in the middle can be added, deleted, merged or split. Additional spots can be added to the map. Our tests have shown that you can have different levels of editing (little or a lot) and still get similar coverage results. It is key to be consistent so that you get similar results across experiments.

Why are spot outlines larger than the spots in 2D mode?

In 2D mode spots may look like they extend beyond the spot boundaries but in fact if you look at them in 3D mode they are in fact perfectly aligned to the background. This video shows the difference in 2D and 3D mode in real time and the ways you can quickly validate the software that you currently use.

Coverage

How is percentage coverage calculated?

Spot coverage is the percentage of spots in the comparison being made which are present on the secondary image. Percentage coverage is calculated against the chosen base image using the calculation:

Percentage coverage = (Common + Additional)/(Common + Missing + Additional) x 100

How does identifying spots as present or absent allow results to be calculated?

The present or absent categorisations from the spot map allow comparisons of images to be made. When any two images (the base and the secondary) are compared the spot presence/absence settings are used to determine which spots are Common, Missing or Additional and are used to calculate percentage coverage.

 

  Base image Secondary image
Common Present Present
Missing Present Absent
Additional Absent Present
Not-Present Absent Absent

See what SpotMap can do for you:

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