PCR tab

When Assay Plugins for multiple devices are stored under the same assay card, don't forget to select the applicable cycler first before you continue on this page.

When an Assay Plugin is configured for multiple PCR cycler/major assay version combinations, the user will need to explicitly select the correct combination to avoid errors when applying changes to the assay configuration. 

Protocol information

This optional field is only available when FastFinder Workflow is enabled and can be used to transfer the PCR Protocol (eg CFX .prcl filename) to the cycler. (eg in the CFX .plrn file creation).

Passive reference dye

In case a Passive reference dye is required by the Assay Plugin, this section is visible.

Dye name

Dye name as defined by the Assay Plugin.

Channel instrument reference

The Channel instrument reference is manual adjustable to aid in the automatic plate setup. See How to automate your PCR set up?

Mix information

Mix name

Mix name(s) as defined by the Assay Plugin.

Mix instrument reference

The Mix instrument reference is manual adjustable to aid in the automatic plate setup for some cyclers. See How to automate your PCR set up?

Target information

Mix name

Mix name(s) as defined by the Assay Plugin.

Target name

Target name(s) as defined by the Assay Plugin. The symbol in front of the target identifies the type of PCR: amplification, end-point or melt.

Target instrument reference

The Target instrument reference is manual adjustable to aid in the automatic plate setup. See How to automate your PCR set up? This field can also be used in the CSV export file to match the target name with the LIMS target code (Target_x_reference).

Reporter dye name

Reporter dye name(s) as defined by the Assay Plugin.

Reporter instrument reference

The Reporter instrument reference is manual adjustable to aid in the automatic plate setup. See How to automate your PCR set up? The data from the reporter dye as set in this field will be taken into account during the analysis in FastFinder.

Quencher dye name

Quencher dye name(s) as defined by the Assay Plugin (if applicable).

Quencher instrument reference

The Quencher instrument reference is manual adjustable to aid in the automatic plate setup. See How to automate your PCR set up? The data from the quencher dye as set in this field will be taken into account during the analysis in FastFinder and can be visualized in the graphs under amplification data.

Standard curve criteria

In case the Assay Plugin allows you to define your Standard curve criteria (manually adjustable), you can configure them in this section. Here, the applicable standard curve criteria per target are shown. Please note that when changing one of the criteria, all existing standard curves will be made invalid with the warning message: 'Standard curve cannot be used anymore: Standard curve criteria have changed.'

The applicable standard curve criteria per equation are shown when clicking on the triangle in front of the list in the Standard curves section (see below)

Standard curves

When your Assay Plugin supports quantitative analyses, this section is displayed. Authorize an analysis first with a valid in-run standard curve to save it and visualize it in a list. Or in case your Assay Plugin supports it, add a standard curve manually.

The Filter button allows you to search and filter on 'Target name' or 'Instrument ID'

Manually added standard curves or standard curves from authorized runfiles will populate this list.

The used standard curve equation during an ex-run quantification analysis, is shown in the PCR setup tab, under 'More info'. By opening the drop-down menu (triangle icon), you can select between other available active standard curves and re-analyze the runfile. This is only available when no quantification standards are already assigned on the plate.

Target name

Target name as defined in the Assay Plugin for which the standard curve applies.

Slope

Slope of the standard curve.

Y-intercept

Y-intercept of the standard curve.

E (%)

Efficiency of the standard curve.

R²

Correlation coefficient (can be N/A in case of manually added standard curves).

Dynamic range

Cq values of the lowest and highest quantification standard. Outside this range, extrapolation can be used to define the quantification of a regular sample (in case enabled in the Assay Plugin).

Valid QS

The number of valid quantification standards. Can be N/A in case of manually added standard curves.

Instrument ID

Instrument ID for which the standard curve applies

Reference name

For automatically generated standard curves, this will indicate the name of the analysis in which the standard curve was created.

For manually added standard curves, this will indicate the name of the manually saved standard curve including an icon indicating the standard curve was manually created.

Created at

Creation date of the analysis run file containing the standard curve or when the standard curve was manually added.

Active toggle

The latest authorized standard curve will be selected by default in the next quantification analysis using ex-run standard curves.

Manually adding a standard curve

When no Standard curve has been previously added, click on the 'Add' button (see above). This will open a flyover screen where all necessary information can be entered. The 'Add' button will only be available when manual creation of standard curves is allowed in the Assay Plugin. 

In case a previous standard curve is already created, the add button is displayed on the right of the screen.

• Instrument: a list of all available instruments will be displayed here. Make sure you have a file authorized from the instrument you want to assign in order to display it here. (See Lab configuration - Instruments).
• Target: select a target from the list, pre-defined by the Assay Plugin.
• Reference name: give a reference name to the Standard curve, for easy identification during PCR setup.
• Slope: Enter a slope between the ranges set in the Assay Plugin.
• Y-intercept: Enter a Y-intercept between the ranges set in the Assay Plugin.
• Dynamic range min: Select the minimum Cq value for which no extrapolation is needed.
• Dynamic range max: Select the maximum Cq value for which no extrapolation is needed.
• R²: Enter a correlation coefficient between the ranges set in the Assay Plugin.

Relative quantification

When your Assay Plugin supports relative quantification, this section is displayed. Here, information regarding the configuration of the relative quantification can be found and adjusted (if assigned the Admin role). Per target, the reference gene is indicated and the corresponding efficiencies (displayed as 'E'). The latter is adjustable (if configured as a lab depending parameter in the Assay Plugin). 

Sample type name

Sample type name as defined in the Assay Plugin.

Type

Type of sample (defined in the Assay Plugin).

Reference sample

Sample that is used as the reference to perform relative quantification. 

Target

Target for which the relative quantification is calculated. 

E target

Efficiency of the target. If configured in the Assay Plugin, this value can be updated by users with an Admin role.

Reference gene

Reference gene which is used for the calculation of the relative quantification. 

E reference gene

Efficiency of the reference gene. If configured in the Assay Plugin, this value can be updated by users with an Admin role.

Lab dependent cutoffs

Only lab dependent (manual adjustable) cutoffs will be shown here. The default cutoff value (as originally configured in the Assay Plugin) and the coupled actions to the executed action when a cutoff is exceeded or failed can be consulted in the Assay Specification document (See Understanding your Assay Plugin: Supportive information about the design, functionality and available features). This document (aka: decision tree documentation, .HTML file) is provided with every Assay Plugin version and can be requested via your Assay provider or via support@ugentec.com clearly stating the Assay Plugin name and version.

When Assay Plugins are created in FastFinder Studio, an overview of all the cutoffs is also shown in the Specification sheet (History - Assay details). (See Configuring and understanding your Assay Plugin).

To change a cutoff/range value: click on the numerical value you want to change, enter the intended value and click on enter. Please note that prior to changing a value, the Assay logic (and consequences of changing a value) is fully understood.

Cq cutoffs

Please find below an example (the correct Decision tree rules are described in the Assay Specification document).

• When a target is detected and has a Cq value lower than 10, an error notification is added to the target result. 
• When the IC target of the Negative Control (NTC) is detected and has a Cq value larger than 32, the target result is set to 'not detected'. 

Baseline corrected end fluorescence (dRnEF) cutoffs

Please find below an example (the correct Decision tree rules are described in the Assay Specification document).

• All detected MS2 IPC targets with a dRnEF below 1000 will be set to 'not detected'.
• All detected N Protein targets with a dRnEF below 0 will be set to 'not detected' - This is an example of an empty Decision tree rule added at the initial development of an Assay Plugin. At a later stage, when you want to implement/use this cutoff, you can change the value directly, without the need to update the Assay Plugin.

Concentration cutoffs

Please find below an example (the correct Decision tree rules are described in the Assay Specification document).

• The sample with a target concentration below 500 will be set to 'invalid'.
• The sample with a target concentration above 200000 will receive an information message to dilute the sample for correct quantification.

RQ cutoffs

Please find below an example (the correct Decision tree rules are described in the Assay Specification document).

• The sample with a relative quantification below 0.37 will receive an information message that the gene expression is considered negligible and the target is set to 'not detected'.

Reference curves

See Analysis - Graphical layout on how to save reference curves.

Saved reference curves from an authorized analysis will populate this list.

In Analyses, the used reference curve during analysis is shown in the PCR setup tab, under 'More info'. By opening the drop-down menu (triangle icon), you can select between all other available active reference curves and re-analyze the runfile. The selected reference curve can be visualised in the graph for comparison reasons during an analysis.

Reference curves within algorithm version 50py or higher

As of Assay Plugin algorithm version 50py (compatible with FF4.12 or higher), it is possible to mark curves as reference curves (as described above) that are taken into account by the algorithm to determine the result states (see remark in screenshot above (orange rectangle)), i.e. addition of historical context. This results in an augmented amplicon group which is then analysed by the algorithm. Especially in runs with all negative or low-context runs, the addition of a reference curve will prove useful. 

Note: To make the most of our algorithm's capabilities, it is essential to use reference curves
that exhibit clear positive curves with shapes similar to those of the samples in the
assay. If you are using artificial control samples as a source of reference curves,
please ensure that these samples produce curve shapes that are appropriate for the
assay. Be cautious using artificial control samples, since artificial control samples
often generate idealized curve shapes, which may differ from the curves observed in
real-world samples

Target (Mix)

Target and mix identification for which the reference curve is saved.

Reference name

By default, the samplename_channelname is chosen but can be changed upon creation for easy identification.

Instrument

Instrument ID from which the reference curve is saved and for which it can be applied.

Created at

Creation date of the analysis file. (not the authorization date when the curve is saved)

Active toggle

Clicking on the triangle in front of a row will open all saved reference curves per target (mix) - instrument combination. By default, the latest saved reference curve will be selected in the next analysis. Marking a reference curve as default will select this reference curve as the default one during the next analysis.

Clicking on the hyperlink icon at the end of the row will open the 'Archive - Target results' filtered on the reference curve for easy access and visualization of the graph and target details.

Cross-contamination detection

Cross-contamination can ruin an entire analysis. FastFinder can be set up to automatically detect cross-contamination. By default this option is disabled. When enabled, the laboratory has the choice to perform the cross-contamination check based on strong and weak Cq cutoffs or during relative Cq values.

It is determined in the Assay Plugin specifications whether or not the extraction plate location is taken into consideration as neighbours as well (in case your laboratory tracks this information using FastFinder Workflow).

Cq cutoffs

If, in a single well, Target X has a Cq < Strong positive Cq and # well(s) surrounding that well have a Cq > Weak positive Cq for the given target, the weak positives will be marked with a cross-contamination notification.

The minimum amount of weak positives and the Cq values to define a strong and weak positive target can be manually adjusted.

Relative Cq values

If, in a single well, Target X has a Cq that is < Delta Cq lower than # well(s) surrounding that well for the given target, the weak positives will be marked with a cross-contamination notification.

Enable the toggle to use relative Cq values. The minimum amount of weak positives and the delta Cq value can be manually adjusted.

Prevalence-driven contamination detection

With these statistics-driven checks, we'll determine whether the number of positives and number/size of positive clusters on your plate is abnormal or not for a specific prevalence. This feature is by default disabled. When enabled, the laboratory has the choice to manually adjust the confidence interval and the prevalence percentage per target.

Other assay settings

Special cutoff requests defined by the customer and developed internally by Velsera will also be visualized in this section.

For example: 

• When the maximum amount of anomalous curves is reached, the results will be made invalid.

Versions

Device identifier

The unique device identification (UDI) as defined in the Assay Plugin is displayed here. A UDI is a unique numeric or alphanumeric code related to a medical device. It allows for a clear and unambiguous identification of specific devices on the market and facilitates their traceability.

Production identifier

Each AP that is made available to the customer, via upload to the FastFinder platform, has a specific version. When an update is made to the AP, the versioning will increase accordingly.

During development by UgenTec, a draft AP is created. Draft APs always are versioned as 0.x. Upon each new iteration, the version will be increased (e.g., from 0.3 to 0.4).  Note that not every draft version is made available to the customer.

Once the customer approves the AP, i.e., provides design freeze, the final draft of the AP will be validated, thereby generating a ‘1.x’ version. In case of a minor update to a validated AP, e.g., minor correction during the validation procedure, the versioning will increase accordingly from 1.0 to 1.1. In case of a major change, e.g., a change request, the AP version will increase from 1.x to 2.x.

When an AP is created with FastFinder Studio, a v1.0 is created directly.

Assay Plugins marked as 'Default' in the General tab will receive a house icon next to their version number.

Intended use

Optional field that states the regulatory intended use as defined in the Assay Plugin: RUO, IUO, IVD, AFNOR, AOAC.

Documentation

It is possible to add either a PDF document or URL link to the Assay Plugin (e.g. assay instructions for use, weblink to support website). This documentation is only available for technically validated Assay Plugins (i.e. not for Assay Plugins created in FastFinder Studio).

Assay label

Label of intended use as provided by the legal manufacturer of the plugin. Assay labels are only available for technically validated Assay Plugins (i.e. not for Assay Plugins created in FastFinder Studio) and is mainly added when the Assay Plugin is registered to the legal authorities (IVD, FDA, ...).

Integration of Assay information in Analyses

Information regarding the Assay Plugin can also be consulted in the Analysis section itself. This information is available to all users (contrary to Lab configuration which is only accessible for Admin users). 

When clicking on the assay name (both in the Summary and Details tab), a flyover screen appears showing information about the Assay Plugin, i.e. version, intended use, owner and documentation. When clicking on the latter, either the PDF file or URL is opened (depending on what is configured).

Furthermore, an overview of the open (i.e. lab depending) cutoff values, standard curve parameters, ... is shown here, visible to all users. Note, the values can only be updated in the Assay settings by Admin users (accessible via Lab configuration).

Finally, (Admin) users can quickly navigate from here to the assay in Lab configuration (by clicking 'Go to assay' at the top). 

 

Relevant articles

Lab configuration - Assays

How to automate your PCR set up?

Understanding your Assay Plugin: Supportive information about the design, functionality and available features