Review the quality data

After the Applied Biosystems™ Analysis Software processes your project, you can use the Data Review screen to review the quality data that are generated by the analysis. The software provides various options to review the quality data; however, the strategy that you use depends on the type of quantification that you are performing and the samples/targets that you are evaluating. The following procedure describes a general approach to data review and provides an overview of the software features.

If you have not already done so, click Analyze to analyze your project.
In the Applied Biosystems™ Software, click Data Review to view the Data Review screen.
From the dropdown list at the top of the screen, select the way that you would like to organize the quality data:
- Targets – Groups and displays the quality data by target name.
- Samples – Groups and displays the quality data by sample name.
- Plates – Groups and displays the quality data by experiment/plate.
Note: The Plates view is common to all real-time instrument software manufactured by Thermo Fisher Scientific .
Review the amplification plots for irregularities and quality flags.

Note: The Applied Biosystems™ Software displays summaries of the quality data in the margin beneath each amplification plot. You can view the identity of any flag by hovering the mouse over the flag of interest.
If flags or irregularities are present, or you would like to review the amplification data for a specific target, sample, or experiment, click the amplification plot of interest to zoom the display.

Click

, then configure the display options for the amplification plot.

Group	Select	Description
Plot Type	ΔRn vs Cycle	Displays ΔRn as a function of cycle number, where ΔRn is the magnitude of normalized fluorescence signal generated by the reporter at each cycle during the PCR amplification. You can use this plot to identify and examine irregular amplification and to view threshold and baseline values for the run.
	Rn vs Cycle	Displays Rn as a function of cycle number, where Rn is the fluorescence signal from the reporter dye normalized to the fluorescence signal from the passive reference. You can use this plot to identify and examine irregular amplification.
	CT vs Well	Displays C_T (C_q) as a function of well position, where C_T is the PCR cycle number at which the fluorescence meets the threshold in the amplification plot. You can use this plot to find outlying amplification (outliers).
Graph Type	Linear	Displays the data on a linear scale.
Graph Type	Log	Displays the data on a logarithmic scale.
Color Type	Well	Colors the data for each well according to its position on the reaction plate.
	Sample	Colors the data for each well according to the sample that it contains.
	Flag Status	Colors the data for each well according to whether it generates quality flags.
	Amp Status	Colors the data for each well according to the amplification status that it is assigned.

If the amplification plot that you are viewing includes data from more than 384 wells, use the histogram beneath the Amplification Plot to view the data of interest:
- Click and drag the anchor icon () to the desired location in the histogram to display the curves from the 384 wells with values nearest to the position of the icon.
- Select the heading of a column in the Well Table that contains numerical content (such as, Amp Score or C_T/C_RT without Flags) to change the x-axis content of the histogram.
Note: Selecting the heading of a column in the Well Table that contains less than 384 data points hides the histogram. The feature is present only when the plot contains more than 384 amplification curves.
If the data set that you are viewing consists of numerous data points, use the Outlier Wheel to organize, filter, and review the data for irregular amplification:
1. In the right-side pane of the Review Plate screen, select View By > Outlier Wheel.
2. From the Sort By dropdown list, select the attribute by which you want the Applied Biosystems Software to filter the displayed data set.
3. While viewing the data set in the Outlier Wheel, select a segment of the wheel to view the associated data in the Amplification Plot and Well Table.
  
  Note: Click the center of the wheel to deselect the data.
4. If desired, click and drag across a segment of the Outlier Wheel to review the related subset of the displayed data.
  As you select and filter the displayed data, the Applied Biosystems Software lists the filters that you apply at the bottom of the Outlier Wheel Plot. To remove a filter, either click Undo to remove the last filter that was applied or click the X next to a desired filter to remove it.
5. At any time while reviewing your data, click Show Table to view the tabular data for the datapoints present in the Outlier Wheel Plot.
See Outlier Wheel Plot for more information on the Outlier Wheel plot.
Review the amplification plots if needed.
When reviewing the amplification data, look for:
- Regular, characteristic amplification of all samples. If irregular amplification is present, consider omitting the individual wells from the analysis.
- Correct baseline and threshold values. If not, consider manually adjusting the baseline and/or threshold values in the analysis settings.
Click Multicomponent to review the multicomponent plot if needed.
When reviewing the multicomponent plot, look for:
- Consistent fluorescence of the passive reference. The passive reference dye fluorescence level should remain relatively constant throughout the PCR process.
- Consistent fluorescence of the reporter dye. The reporter dye fluorescence level should display a flat region corresponding to the baseline, followed by a rapid rise in fluorescence as the amplification proceeds.
- Irregular fluorescence. Spikes, dips, and/or sudden changes in fluorescence should not be present.
- No amplification in negative control wells. Negative control well should not amplify. y

View and modify the data in the Well Table.

Tool	Use this tool to
Mouse/cursor	To select: An individual well, select the well in the Well Table. More than one well at a time, Ctrl+ or Shift+click the wells in the Well Table. When you select wells in the Results table, the corresponding data points are selected in the amplification plot.
Group By dropdown list menu	Select how to group the samples in the Well Table. For example, if you select Target, the samples are grouped according to the nucleic acid sequence they target.
Actions dropdown list	Bookmark/Clear Bookmark for wells in the project. The bookmarks persist in the Data Review and Results screens to easily identify bookmarked wells.
Actions dropdown list	Omit/UnOmit well from the analysis. After you omit or unomit a well, click Analyze to reanalyze the project. For omitted wells, the software: Does not display data or tasks in the Well Table. Does not include the omitted wells in the analysis. For unomitted wells, the software reassigns the tasks based on the settings in the Analysis Settings dialog box.
Flag Details	Select Show Flag Details to display the results of each quality flag in an individual column. When unselected, the table displays the results of the quality analysis in a single column.
or	Expand or collapse the Well Table.

Review the data in the Well Table data.

Column	Use this column to
Well	View the location of the well in the reaction plate. For example, P18 indicates that the sample is in row P, column 18.
Sample	View the ID (a unique name or number) of the sample.
C_T /C_RT	View the C_T /C_RT calculated for the related well.
C_T /C_RT (Post IC)	(Inter-plate calibrator analysis only) view the C_T /C_RT calculated for the related well following inter-plate calibrator (IC) normalization.
Eq. C_T /C_RT	View the equivalent C_T /C_RT calculated for the related well. Note: The Eq. C_T Mean values are equal to the C_T Mean values in the Results Table of the Analysis tab.
Eq. C_T /C_RT Mean	View the mean equivalent C_T /C_RT calculated for the related well. Note: The Eq. C_T SE values are equal to the C_T SE values in the Results Table of the Analysis tab.
Eq. C_T /C_RT SE	View the standard error (SE) of the equivalent C_T /C_RT calculated for the related well.
(Bookmark)	View whether or not the well has been bookmarked.
Omit	View the omission status of the related well.
Amp Status	View the amplification status as determined by the software, where possible states are: Amp – Target amplified. No Amp – Target did not amplify. Inconclusive – Unable to determine if amplification occurred. Review run data. N/A – The well was omitted from analysis or Insufficient cycle number to determine if amplification occurred.
Amp Score	View the amplification score calculated for the well.
Cq Conf	View the confidence value calculated by the software for the C_Q (C_T) for the given well.
Quantity	View the calculated quantity for the given well
Target	View the ID (a unique name or number) of the nucleic acid sequence targeted by the assay added to the well.
Plate	View the barcode of the reaction plate used to run the reaction. If no barcode is present, the software displays the name of the experiment file to which the data belongs.
Baseline Start	View the start and endpoints of the range of PCR cycles used as the baseline in the calculation the C_T for the related well.
Baseline End
Task	View the task assigned to the well. A task is the function that a sample performs: Unknown No template control (NTC) (control identifier)
Flags	View the number of flags generated for the well.

When ready, click to return to the QC thumbnails.
Review the amplification data for specific targets, samples, or experiments if needed.
Click in the toolbar to review the quality summary.
Review the Quality Summary for any flags that the project data generates. For each quality flag, the table displays the number of times that the flag was triggered by the project data. To examine the data that triggered the flag, click the link in the Name column to view the amplification data for the related target, sample, or plate.

In response to the presence of quality flags, consider the following resolutions:
- Change the quality settings in the analysis group:
  - Adjust the sensitivity of the quality flags so that more wells or fewer wells are flagged.
  - Deactivate the quality flags that triggered by the data.
- Omit individual wells from the analysis.

Contents

Index

Glossary

Search Results

Review the quality data