Analysis - PCR setup

In the tab PCR setup, users can manually assign the desired AP to an analysis, or make adjustments to the plate configuration. Different sample types or mixes can be assigned if needed. To apply the updated configuration to the runfile, reanalysis is required.

Plate configuration

On the left side, the plate configuration is visualized. Here, the plate is shown, together with the details of the samples that are present.

At the top, the PCR instrument type is visualized together with the instrument’s ID.

The well selection can be done by clicking on the desired well, either on an individual basis or multiselect (press and hold Ctrl button or drag your selection over multiple wells). Alternatively, it is possible to make use of the designated buttons situated above the plate lay-out:

Symbol	Meaning
	Single selection pattern
	Column selection pattern
	Row selection pattern
	Quadrant selection pattern
	Select all wells
	Deselect all wells

 

By selecting these patterns, the full column/row/quadrant will be automatically selected, when a single well is selected.

If applicable, replicate groups can be created and/or removed. To visualize the replicates, the button ‘View replicates’ needs to be toggled on: . Wells that are not assigned to a replicate group, will not have any numbering listed in the well. The numbering style will follow the plate setup numbering, i.e. letter + number for rectangular plates (A1, B1, C1) and number-only for rotor plates (1, 2, 3).

Replicate groups can only be formed in case the sample name, mix, and sample type and/or concentrations correspond. In case the Assay Plugin supports replicates, the decision tree will take this into consideration.

To create a new replicate group, the desired wells need to be selected, followed by clicking on 'Actions' - ‘Create replicate group’ button () (Figure, left). In a similar manner, wells can be removed from a

replicate group, by selecting the desired wells and choosing the option ‘Remove selection from replicate’ () under 'Actions' (Figure, right).

Alternatively, it is also possible to automatically detect and assign replicates on the right side.

For quantitative analyses, if enabled in the Assay Plugin, certain adjustments can be made to the quantification configuration. Via 'Actions' - ‘Edit quantification configuration’, a fly-over screen is opened where the user can:

• enter the correct concentration values for the quantifcations standards (User-defined concentrations).
• compensate for dilutions and other corrections when calculating the sample concentration (Quantification configuration - Correction factor).
• prevent unrequested quantified results from being reported (Quantification configuration - Restrict quantification).

Assign assay

On the right hand side of the PCR setup tab, assignment of the correct assay can be done, if not done automatically. When no AP has already been assigned to the analysis, an overview of all the possible assays (based on the available targets) is shown. Using the search bar, one can perform a search based on the name to quickly find the desired AP. If an AP has already been assigned, the assay card will indicate ‘Assigned’. By toggling the button ‘Show assigned only’ off, again an overview of all possible APs is provided (without losing the current assignment or plate configuration).

If the PCR setup has not been done automatically, an overview of all APs that can be assigned to the plate is shown. The correct AP can be chosen from this overview and the sample types can be assigned. To assign the correct sample type to a specific well, the user can do the following:

1. Select the well(s) (to select multiple wells, hold the Ctrl button) and click on the correct sample type icon in the Assay card, or
2. Drag and drop the sample type icon from the assay card to a specific well.

If the nametags are correctly set up, it is also possible to select the well, a subset of wells or the entire plate and click on the ‘?’ icon from the Assay card. This will automatically assign the correct sample type to the specific locations.

Following actions are possible:

• Automatic replicate detection ()
  When replicates are present on the plate, they can be easily identified using this function, rendering manual assignment unnecessary.
• Automatic assignment ()
  When a new version of the AP becomes available, it is possible to quickly assign this version to the current plate set-up and rerun an analysis that already has been done.
  Updating the plate set-up using ‘automatic assignment’ should work under the following circumstances:
  • If a new AP version becomes available next to the previous one (i.e., new assay card in Assay overview). The old version (assay card) needs to be inactivated.
  • If a new AP version becomes available on top of the previous one (i.e., no new assay card in Assay overview). The old version does not need to be inactivated. However, we always recommend to inactivate older versions to prevent confusion.
• Add existing assay ()
  Users are redirected to Lab configuration - 'Add new assay' to enable direct addition of a new assay plugin. From here, it is possible to import a new AP (version) when available. When the assay is added, the user is directed back to the PCR setup tab.
• Add new assay () (optional)
  If Studio is enabled and depending on the instrument, users can directly start with the creation of a new Assay Plugin based on the runfile that has been uploaded. From here, Studio is directly opened, containing prefilled information about the assay. This feature is only available for the following instruments: CFX, ABI7500, Quantstudio 3, 5, 5Dx, 6 Flex/Pro, 7 Flex/Pro, 12K Flex and ViiA7. 

When an analysis is uploaded for any of the abovementioned instruments, but there does not exist an Assay Plugin yet, users with access to Studio can start the configuration of the Assay Plugin from the PCR setup tab ('Create new assay').

A fly-over screen will open displaying the mixes, dyes and targets that are automatically recognized from the runfile. The user can then enter the Assay name, select the PCR mixes that need to be included (and if applicable, select which target is an internal control). Target orders can be re-arranged from this screen as well. From here, the user is redirected to Studio ('Go to Studio') where the configuration of the Assay Plugin can be finalized. When the Assay Plugin is published, the user returns to the analysis and click 'Add existing assay'. This will open the Lab configuration screen, where the newly created Assay Plugin can be imported into FastFinder. The Assay Plugin is then automatically assigned to the analysis. 

When an assay has been assigned, this is visualized by the Assay card (with the addition ‘Assigned’ in the header). Furthermore, more information about this assay is available when clicking on the button ‘More info’ which will unfold a dropdown overview. Following information is shown (if applicable):

• Versions
  Overview of the current (and if present previous) version(s). Only older versions published under the same assay card will be shown. For these versions, it will also be visible whether they are still active or not.
• Color compensation
  If applicable, the color compensation file (CC) assigned to this assay is shown. All available CC files are available when clicking on the triangle
• Standard curves
  Overview of standard curves that were manually created.
• Reference curves
  Overview of available reference curves per target (as saved in previous analyses).

Once everything is set up properly in the PCR setup tab, the user can click on the ‘Analyze’ button (top right corner) to perform the analysis. If the automatic plate setup has been properly configured, the analysis will be run automatically and the user is guided directly to the Summary tab.

During the analysis review, it is always possible to make alterations to the PCR setup. From the Summary or Details tab, the user needs to switch (back) to the PCR setup tab and make the necessary changes. Next, the user needs to click on the ‘Reanalyze’ button to reanalyze the runfile with the updated configuration.

Finally, the user can always perform a number of actions during the analysis. When opening the drop-down menu by clicking on the ‘Actions’ button (right top corner), the same actions as found on the Analyses overview are available:

• Assign label
• Assign user
• Discard analysis

More detailed information about these actions can be found at the Analyses - Overview page. It is also possible to directly assign a label and/or assign a user to the current analysis by clicking on the second and/or third button, respectively, next to the analysis name.

Finally, a user can access certain audit trail information regarding a specific analysis by clicking on the text balloon icon on the lefthand side. This will open a pop-up screen where information can be found. This includes who performed which action at what moment.

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