In the Coverage step you can explore and edit your coverage results with dedicated tools. The default screen displays the coverage for (one of) your coverage pair(s). But you can examine coverage for additional image pairs, and display a summary of all your results.
By default, you will see two or more tabs at the top of the screen:
- A Coverage summary tab, where you can create and manage additional pairs for coverage analysis. It also summarizes the coverage results for selected coverage pairs.
- A tab for the coverage analysis of the current image pair.
- A tab for the coverage analysis of additional image pairs, if your experiment includes at least two secondary images.
There will be an additional tab for each newly opened coverage analysis. Clicking a tab will show the coverage results for the corresponding image pair, organized into the following screen areas.
The Display area shows the two images of your image pair. In principle:
- The primary image, at the left, represents the total protein content of the HCP-antigen.
- The secondary image, at the right, reveals the proteins detected with the anti-HCP antibody.
At the right of the Display area, you will find the standard Toolbar to manipulate the image views, and set the visualization options.
The spots on these two images are color-coded according to their coverage Status (see below).
The Coverage table lets you view and edit a spot’s coverage Status as a colored square. The software automatically assigns this status based on the output of the presence filter in the previous step. Indeed, based on the quantitative filters, every spot was assigned a State on each image:
The spot’s state on the primary image is displayed in the P:State column in the Coverage table (P for Primary image). Similarly, the spot’s state on the secondary image is displayed in the S:State column (S for Secondary image). The table also displays the spot’s Intensity and Volume for each image.
Manually edited spots are an exception to the above rules. Such spots are always showing a default uncertain status, regardless of the spot’s filter states.
All spots with a yellow component in their coverage status are displayed as uncertain (yellow) on the images and in the Coverage diagram.
The default coverage status can be reviewed and edited.
Melanie uses the status of all spots to calculate the Coverage for your image pair, i.e. the percentage of immunodetection that the antibody reagent offers for the total population of HCPs. In the Coverage tab of the General Options, you can choose how Melanie calculates the coverage (for each of the three possible coverage types):
- Intersection vs Primary image spots:
Coverage = (Common) / (Primary + Common)
- Secondary vs total number of spots:
Coverage = (Common + Secondary) / (Primary + Common + Secondary)
- Intersection vs total number of spots:
Coverage = (Common) / (Primary + Common + Secondary)
You can visualize the coverage for your image pair, including its calculation, using one of the following options:
The coverage is tentative as long as there are uncertain spots. When you review the uncertain spots and assign them an appropriate status, percent coverage will be updated.
All three coverage diagrams are interactive. You can click on the numbers in the colored areas to select the corresponding spots on the images and in the Coverage table. Clicking on one of the image names at the top of the diagram selects all the spots considered present on that image.
The Review status section offers various options and tools to simplify review of coverage status.
Choose which spots you want to visualize on the images, by selecting an option in Show spots that are:
- All – Shows all spots
- Qualified – Only shows spots with Primary, Common or Secondary status
- Unqualified – Only shows spots with Uncertain status
- On both images – Only shows spots with Common status
- On primary image – Only shows spots with Primary status
- On secondary image – Only shows spots with Secondary status
- Product – Only shows Product spots
Your choice will determine what spots are shown on the images and in the Coverage table. This is useful when reviewing Unqualified spots for instance.
- Show coverage table – Show or hide the coverage table
- Show targeted coverage table – Show or hide the targeted coverage table
- Select spot set – Select all spots from a given spot set
- Select annotated spots – Select all spots from a given annotation category
- Reset spot qualification – This will reset all status assignments to the default (based on Presence filter output). All user edits of coverage status will be lost.
- Swap images for coverage calculation – This will calculate coverage for the same image pair, but considering the right image as the Primary image, and the left image as the Secondary image. The image positions or colors will not be switched, and the coverage diagram will remain the same. Only the percent coverage and its formula will change. You will be made aware of the switch with the * that will appear after the coverage.
Click the Edit coverage status icon to quickly enter the Coverage mode. You can then edit the coverage status by simply clicking on a spot, both in 2D and 3D view. You can also activate the Coverage mode via the Toolbar.
If you used two thresholds in your presence filters, you will have a number of spots that have been tagged uncertain. You can quickly review these spots to assign them an appropriate coverage status and allow calculation of a final percent coverage. The number of uncertain spots to be reviewed appears in yellow in the Coverage diagram.
When reviewing a spot’s coverage status, you must select one of the four options:
- Primary – The spot is only present on the primary image
- Secondary – The spot is only present on the secondary image
- Common – The spot is present on both images
- Absent – The spot is absent from both images
To prepare for efficient coverage status review:
- Show only Unqualified spots in Review status, so you can concentrate solely on the uncertain spots.
- Activate the Coverage mode.
- Change to 3D view, because this will allow you to better judge whether a spot is present on an image or not.
Use a mouse with a scroll wheel to avoid frequent mode changes to move, zoom and rotate images or increase peak height in 3D view. You will appreciate the simplicity and time savings of using the scroll wheel to manipulate your images.
The default peak heights in the 3D view may give you the impression that many spots are absent. When you increase the peak height however, you will often find that there is a real protein signal. So it is important to adjust the peak height properly. At any moment, you can increase or decrease the peak heights in the 3D view by rolling your mouse scroll wheel while holding the Ctrl key.
You can review and edit coverage status in two ways:
- Click on a spot in one of the images and then select the appropriate coverage status from the four-color tile. You can select several spots by drawing an area over them, or by using the Shift or Ctrl keys. The chosen coverage status will be applied to all selected spots. The spot(s) remain(s) selected and visible until you select the next spot.
- Select a spot in the Coverage table, click on the colored square and choose the appropriate coverage status from the four-colored tile. Once this is done, the spot disappears from the table and the image, and the next spot will be selected for review.
After reviewing the uncertain spots, it is good practice to do a rapid visual inspection of all spots on your images. Indeed, when spots contain artefacts or are partly overlapping with other spots, their spot value (intensity or volume) may be exaggerated. As a result, some spots may have been labeled present whereas they are absent in reality. On the other side of the spectrum, spots that are saturated cannot be correctly quantified. They can have spot values close to zero and therefore be labeled absent.
To do this quick check on all spots, show All spots in Review status. If you find obvious inconsistencies, you can edit the coverage status as explained above.
All coverage pairs
Once you reviewed your current coverage pair, you can switch tabs to review coverage status on your other image pairs as well.
Instead of calculating coverage for all spots in your images, you can look at the coverage for a specific subset of spots. If you defined molecular weight (MW) markers for instance, you could use Filter by values in the Coverage table to select spots above and/or below a certain MW and create coverage targets from them. You can then assess whether your antibody is sufficiently reactive, not only against high MW spots, but also against low MW spots.
In the example below, coverage for all spots was 57%, but for spots with a MW under 25000, it was only 34%.
Use the icons in the Targeted coverage table to manage your targets:
Create targeted coverage – Define a new target (selection of spots) for coverage calculation. You can:
- Create targeted coverage from spot selection, if you have spots selected.
- Create targeted coverage from spot set, if you previously defined a spot set.
Remove targeted coverage – Remove existing targets by selecting them from a list.
Spot selection, manually or via Filter by values, only acts on spots shown on the images and in the Coverage table. So when selecting spots, make sure to show All in Review status. While absent or uncertain spots are not considered in the coverage calculation, it is better to include them in your selection. This way, they will be taken into account in case you change their status.
When you select a target in the Targeted coverage table, only the spots from this target are shown on your images. And the Coverage diagram now shows the coverage for the selected target.
The Coverage summary table in the Coverage summary tab shows a list of one or several coverage pairs. If you only have 2 images in your project, you will see a single image pair (row) and no additional pairs can be created. If you have more than 2 images in your project, there will be one pair (row) for each secondary image against its corresponding primary image. But you can create and manage additional coverage pairs. You may, for instance, compare the two primary images, or the two secondary images.
Click New to create an additional coverage pair. Select one or several pairs (hold the Shift or Ctrl keys) from the Add new coverage pairs to compare window. The new coverage pairs open in new tabs and will be added to the Coverage summary table.
To delete a coverage pair, select it in the Coverage summary table and click Delete.
Click Properties to view and edit the Name and Note of a selected coverage pair.
Click the Options icon at the top right of the Coverage summary table and then choose Column settings. Tick all the fields to take a look at the information you can display:
- Name – The name of the coverage pair. You can edit this name by clicking the Properties button.
- Open – Tick or untick this box to show/hide your coverage pair from the Coverage summary histograms and the tabs.
- Primary image, Secondary image – The names of the primary and secondary images.
- Primary type, Secondary type – The types of the primary and secondary images, as they were defined in the Quality control step.
- Coverage – The coverage, on a colored background.
- Percent – The coverage, expressed as a bar proportional to the cell width, for a quick coverage comparison between pairs.
- Spot count – The spot counts as bars proportional to the cell width. The included spot statuses depend on the formula used to calculate coverage.
- Spots – The total number of spots considered in the coverage calculation. It equates the denominator of the formula used to calculate coverage.
- Primary, Secondary, Common, Uncertain – The number of primary, secondary, common and uncertain spots.
- Coverage type – The coverage type of the pair.
- Formula – The formula used to calculate coverage. You can choose it in the Coverage tab of the General options, for each of the three coverage types.
- Note – You can add a description of your coverage pair by clicking the Properties button.
Each pair has one of the following Coverage types:
- Primary Secondary – Compares a secondary image (e.g. anti-HCP antibody) against a primary image (e.g. HCP antigen). The default pairs created are of this type.
- Primary – Compares two primary images (e.g. two HCP antigen replicates).
- Secondary – Compares two secondary images (e.g. two anti-HCP antibody images).
You will likely want to use different coverage formulas for these coverage types. Whereas the options Intersection vs Primary image spots or Secondary vs total number of spots make most sense for Primary Secondary coverage types, the option Intersection vs total number of spots is better suited for Primary or Secondary comparisons.
In the Coverage tab of the General options you can specify, for each coverage type, what formula must be used to calculate coverage.
Present your coverage results
The Coverage summary histograms will show the results for all coverage pairs ticked Open in the table. You can personalize how the histograms are presented. To do so, click the Options icon at the top right of the Coverage summary histograms and then choose Display. The following options are available:
Specify whether the vertical axis should display Percent or Spots. You can also change this setting on the axis itself.
|Only percent||Thick percent – thin spots||Equal percent – spots||Equal spots – percent||Thick spots – thin percent||Only spots|
By default, the histograms show vertical bars. You can deactivate the option to show horizontal bars.
By default, the histograms show labels (percent and spots). You can deactivate the option to remove the labels.
At the top of the Coverage summary tab, you can choose the target for which you want to display the coverage results from the Targeted coverage drop down. This will update the table and histograms to display the values for the chosen target. This selection is synchronized with the targeted coverage selection in the other tabs.