QUICK REFERENCE

Mutation Detector™ Software

Publication Part Number 4467104 Rev. B Revision Date April 2012

Contents
■ Well Data table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
■ Replicates Average table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
■ Assay Attributes table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
■ Results table. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
■ Detection ΔCT Cutoff Calculations table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Purpose of the Mutation Detector™ Software

• Import instrument results files (*.csv or *.txt files) from TaqMan® Mutation Detection Assay
experiments that were run and analyzed on supported Applied Biosystems® real-time PCR
systems
• Perform mutation detection analyses to determine the:
– Mutant allele assay detection ΔCT cutoff values for your sample type
– Presence or absence of a mutation
– Percent mutation in samples when a calibration ΔCT value is available
Well Data table
The Well Data table displays well-level data for all reaction plates included in the study.

Column Description Edits allowed?

Plate The reaction plate name No
Note: If no reaction plate name is provided in the instrument results file, the *.csv or *.txt file name is used.
Well The well number from the reaction plate No
Assay The name of the assay in the well No
Sample The name of the sample in the well No
Control (If applicable) The control assigned to the well: NTC, POSITIVE, or NEGATIVE Yes
If you change the control status of a well, you must reanalyze the data to account for the change. Click
Perform Analysis.
Note: IPCs are assigned according to the instrument results files exported from the real-time PCR
system software. If you included IPC reagents in your PCR reactions (from the TaqMan® Mutation
Detection IPC Reagent Kit), the Mutation Detector™ Software simultaneously analyzes the CT values
in the reaction well for the mutation detection assay reaction (FAM™ dye) and the IPC reaction
(VIC® dye). IPCs cannot be edited in the Mutation Detector™ Software.
Sample Ct The CT value of the sample No
IPC Ct The CT value of the IPC No
Quantity The numeric value amount of sample in the well (typically in reference to ng amounts or number of copies). No
Omitted The omit status of the well Yes
When the checkbox is selected, the well is omitted from the analysis. The Well Flag column contains a flag
that explains why the well was omitted.
If you change the omit status of a well, you must reanalyze the data to account for the change. Click
Perform Analysis.
Well Flag A flag that explains why the well was omitted from the analysis No
In the Well Data table, the software assigns only one flag per well. The flag hierarchy is: NOFAM, ACTEC,
IPCEC, OFILE, CTOUT, OUSER.
 Flags in the Well Data table

Flag Description
NOFAM The well contains VIC® dye CT values, but no assay FAM™ dye CT values are detected.
ACTEC The FAM™ dye CT value for the assay exceeds the cutoff value.
IPCEC Both the VIC® dye CT value for the Internal Positive Control and the FAM™ dye CT value for the assay exceed the cutoff
values.
OFILE There was an omitted well in the instrument results file (*.csv or *.txt file).
CTOUT The software omitted a CT outlier in a replicate set.
OUSER A user omitted the well in the Mutation Detector Software.

Assign or change a control

1. Click the Well Data tab.
2. For the well that you want to change, click inside the Control column cell.
3. Select NTC, POSITIVE, or NEGATIVE from the drop-down list. All related replicate wells
are automatically populated with the same control name. The controls are defined in the table
below.

4. Click to reanalyze the data.

Column Description
NTC No Template Control
By default, samples that were named NTC in the instrument results files exported from the real-time PCR
system software are labeled as NTCs in the Mutation Detector™ Software. However, you can change the
NTC assignment in the Mutation Detector Software.
POSITIVE A positive control is an assay run on a sample containing 100% on-target templates (i.e.,gene reference
assays run on a wild type sample or mutant allele assays run on a sample that contains 100% mutant allele).
There are two uses for positive controls:
• Test for proper assay amplification of the assay target. If the positive control fails the CT cutoff value, all
wells with that assay are excluded from mutation detection calculations.
• Real-time calculation of calibration ΔCT values.
NEGATIVE A negative control is a mutant allele assay run on a sample containing 100% wild type allele. Negative
controls are used for real-time calculation of detection ΔCT cutoff values. Note the following:
• When a mutant allele assay negative control sample is selected, all wells containing other mutant allele
assays run on the same sample are automatically selected as Negative controls by the software.
• If a mutant allele assay negative control sample is selected, and a gene reference assay was run with
the same sample, then the software automatically calculates a detection ΔCT cutoff value for the mutant
allele assay. If a calibration ΔCT value for the assay pair is available, then this value will be used to
calculate a normalized detection ΔCT cutoff value for the mutant allele assay.
• You cannot assign negative controls to wells that contain gene reference assays.

Replicates Average table

The Replicates Average table displays the sample-level data for all reaction plates included in the
study. The table below describes each column in the Replicates Average table.
Note: You cannot edit the data in the Replicates Average table.
Note: Technical replicates are not required for mutation detection analysis, but we strongly
recommend that you use technical replicates for real-time calculation of detection ΔCT cut-off
values.
 Column Description
Plate The reaction plate name
Note: If no reaction plate name is provided in the instrument results file, the *.csv or *.txt file name is used.
Assay The name of the assay run with the sample
Sample The sample name
Quantity The numeric value amount of sample in the well (typically in reference to ng amounts or number of copies)
Control The control assigned to the sample
#Replicates The number of technical replicates
The software determines the number of technical replicates by counting all wells on the same reaction plate
that have the same sample name, assay name, and DNA quantity (if not provided, the DNA quantity is
assumed to be equal).
#Valid Replicates The number of valid technical replicates
The software determines the number of valid technical replicates by counting only those wells that have
not been omitted from the analysis. View the Well Data table Omitted and Well Flag columns to discern
which replicate(s) have been omitted and why.
Avg Ct The average CT value for the valid technical replicates
Std Dev Standard deviation of the valid technical replicates
Omitted Samples that have been omitted from the analysis because all wells for the sample were omitted
To include omitted wells in the analysis, you can:
• Change the omit status in the Well Data table.
• Edit the appropriate analysis settings in the Current Study Settings tab.
Flag A flag that explains why the sample was omitted from the analysis
Note: Multiple flags may appear for a sample if more than one flag applies.

Flags in the Replicates Average table

Flag Description
NTCCT The assay CT value for the No Template Control is below the cutoff value (amplification was detected).
PCTEC The assay FAM™ dye CT value for the positive control sample exceeds the positive control CT cutoff value.
RCTOR The FAM™ dye CT value for the reference assay is outside of the defined range.
NTYPE The assay type is unknown. The assay name does not have the correct format or is not specified in the Assay
Attributes file.

Assay Attributes table

The Assay Attributes table displays the predetermined and (if applicable) the real-time calculated
calibration ΔCT values and detection ΔCT cutoff values that are used in the mutation detection
calculations. The table below describes each column or button in the Assay Attributes table.

Column/button Description Edits allowed?

Mutant Assay The name of the mutant allele assay No
GeneRef Assay The name of the gene reference assay that is paired with the mutant allele assay No
Calibration ΔCt(-gr) The predetermined calibration ΔCT values for paired mutant allele assays and gene Yes
reference assays
Cal.Calibr. ΔCt(-gr) The calculated calibration ΔCT values for paired mutant allele assays and gene No
reference assays, when the required positive control data for the calculation exists in
the current study

Mutation Detector™ Software Quick Reference 3

 Column/button Description Edits allowed?
Detection ΔCt Cutoff The predetermined detection ΔCT cutoff values Yes
The cutoff value is the limit of detection that is used to determine the presence or
absence of a mutant allele in a sample. For assays having calibration ΔCT values and
therefore normalized detection ΔCT cutoff values, the detection ΔCT cutoff values are
converted to % in the Results table.
Cal. Detection ΔCt Cutoff The calculated detection ΔCT cutoff values, when the required negative control and No
gene reference assay data for the calculation exists in the current study.
The cutoff value is the limit of detection that is used to determine the presence or
absence of a mutant allele in a sample. If you select real-time calculated detection ΔCT
cutoff values, these values are shown in the results table. If a calibration ΔCT value is
available for the assay, then these values are converted to % in the Results table when
the ‘Show % Mutation’ option is selected.
Reset button Resets all edited values in the table to the predetermined values previously used in the N/A
current study
Import button Imports a custom Assay Attributes file that contains user-defined predetermined N/A
values
Default button Restores all edited values in the table to the default predetermined values from the N/A
'TMDA_default_assay_attributes.txt' file stored in your Mutation Detector Software
installation folder.

Results table
Each row in the Results table displays the analysis results for a sample run with a mutant allele
assay paired with a wild type allele assay or a gene reference assay. The software compares the
calculated normalized ΔCT value to the detection ΔCT cutoff value for the mutant allele assay. The
table below describes each column in the Results table.
Note: You cannot edit the data in the Results table.

Column Description
Plate The reaction plate name
Note: If no reaction plate name is provided in the instrument results file, the *.csv or *.txt file name is used.
Sample The sample name
Quantity The numeric value amount of sample in the well (typically in reference to ng amounts or number of copies)
Assay The name of the mutant allele assay
Detection Cutoff The detection cutoff value for the mutant allele assay, expressed as expressed as a ΔCT value (from the
Assay Attributes table)
The detection cutoff value is expressed as a percentage for assays having calibration ΔCT and normalized
detection ΔCT cutoff values, when the 'Show % Mutation' analysis option is selected.
% detection = 1/2 × 100%
The software allows a minimum % detection cutoff value of 0.001%. The software does not limit the
maximum detection CT cutoff value used for a ‘yes/no’ mutation detected answer.
Note: A detection ΔCT cutoff value is required for mutation detection analysis. If no detection ΔCT cutoff
value is provided in the Assay Attributes table for the analysis, then the mutation detection calculation
cannot be performed, and a NOCUT flag is displayed in the results table.

4 Mutation Detector™ Software Quick Reference

 Column Description
Detected?/%Mutation Detected? – The calculated mutation detection results
Note: Select/deselect The results are displayed as follows:
the Show % Mutation • Y = yes; the amount of mutation detected is below the mutation detection cutoff ΔCT value and/or exceeds
button to toggle between the percent mutation detection cutoff value for the mutant allele assay. That is, the sample ΔCT value
the Detected? view and is less than the detection ΔCT cutoff value.
the % mutation view. The
• N = no; the sample is either mutation negative, or below the limit of detection for the mutant allele assay.
Show % Mutation option
That is, the sample ΔCT value is greater than the detection ΔCT cutoff value.
is available only for
assays with calibration • I = invalid result; a flag or flags indicating the reason for the failure are displayed in the Flag column.
ΔCT values. % Mutation – The calculated amount of mutant allele within a mutation-positive sample for assays with
calibration ΔCT values. Quantitative analysis is not an option for mutant allele assay and gene reference
assay pairs that have not been calibrated.
For a sample in which a mutation was not detected, the software does not calculate a % mutation, but
assigns 0%.
#Valid Replicates (mu) The number of valid technical replicates for the mutant allele assay
The software determines the number of valid technical replicates by counting only those wells that have
not been omitted from the analysis. View the Well Data table Omitted and Well Flag columns to discern
which replicate(s) have been omitted and why.
Avg Ct (mu) The average CT value of the valid technical replicates for the mutant allele assay
Std Dev (mu) Standard deviation of the valid technical replicates for the mutant allele assay
Ref Assay The name of the wild type allele assay or gene reference assay that is paired with the mutant allele assay
for mutation detection analysis
#Valid Replicates (rf) The number of valid technical replicates for the wild type allele assay or gene reference assay
The software determines the number of valid technical replicates by counting only those wells that have
not been omitted from the analysis. View the Well Data table Omitted and Well Flag columns to discern
which replicate(s) have been omitted and why.
Avg Ct (rf) The average CT value of the valid technical replicates for the wild type allele assay or gene reference assay
Std Dev (rf) Standard deviation of the valid technical replicates for the wild type allele assay or gene reference assay
ΔCt The ΔCT value of the mutant allele assay minus the wild type allele assay or gene reference assay in this
sample
Calibration ΔCt The calibration ΔCT value of the paired mutant allele assay and wild type allele assay or gene reference
assay (from the Assay Attributes table) that is used to calculate the normalized ΔCT value
ΔCt norm Normalized ΔCT value (the sample ΔCT value minus the calibration ΔCT)
The normalized ΔCT value is compared to the detection ΔCT cutoff value for the mutant allele assay to
determine sample mutation status.
Flag Flags that explain why a valid mutation detection result could not be calculated for the sample
Note: Multiple flags may appear for a sample if more than one flag applies.

Flags in the Results table

Flag Description
MUPOS A mutant allele assay run on a 100% mutant allele sample is selected as a positive control in the Well Data table.
Note: The MUPOS flag appears when the corresponding assay samples are selected as positive controls in the Well
Data table.
NOREF There is no wild type allele assay or gene reference assay that pairs with the mutant allele assay for the same sample
on the plate.
NOCUT No Detection ΔCT cutoff value (predetermined or real-time calculated) is available in the Assay Attributes table for
the mutation analysis.

Mutation Detector™ Software Quick Reference 5

 Flag Description
Additional flags After the software performs the analysis, all flags relevant to the mutation detection calculations are shown in the
Results table, including:
• Replicates Average table flags, except NTYPE (see page 3)
• Well Data table flags, if all wells for a sample were omitted (see page 2)

Detection ΔCT Cutoff Calculations table

The Detection ΔCT Cutoff Calculations table is dynamically displayed when a detection ΔCT cutoff
value determination experiment is included in the Current Study. Each row in the ΔCT Cutoff
Calculations table displays all the data used to determine a ΔCT cutoff value. In a typical
experiment, three or more wild type gDNA samples are run with mutant allele assays (negative
controls selected in the Well Data table) and paired gene reference assays. The software calculates
a mutant allele assay ΔCT cutoff value when a calibration ΔCT value is not available and a
normalized ΔCT cutoff value when a calibration ΔCT value is available. The table below describes
each column in the ΔCT Cutoff Calculations table.

Flag Description
Assay The name of the mutant allele assay.
Sample The wild type sample name(s). Multiple values are separated by commas.
CT(mu) The CT value(s) for the mutant allele assay (from the Replicates Average table). Multiple values are separated by
commas. For any CT values that are undetermined or greater than the Maximum CT cutoff value (set in the Current
Study Settings), the Maximum CT cutoff value is shown and is used for the ΔCT cutoff calculations.
Ref Assay The name of the gene reference assay that is paired with the mutant allele assay for the detection ΔCT cutoff
determination analysis.
CT(ref) The CT value(s) for the gene reference assay (from the Replicates Average table). Multiple values are separated by
commas
ΔCT The ΔCT value(s) of the mutant allele assay minus the gene reference assay in this sample. Multiple values are
separated by commas
Ave ΔCT The average ΔCT value for all wild type samples run with the same paired mutant allele assay and gene reference
assay
Std Dev Standard deviation of the sample ΔCT values
ΔCT Cutoff ΔCT = [CT(mutant allele assay negative control) – CT(gene reference assay)]– (3 × the standard deviation or 2 CT, whichever
is greater)
Calibration The calibration ΔCT value of the paired mutant allele assay and gene reference assay (from the Assay Attributes table),
if available, that is used to calculate the normalized ΔCT value
ΔCT Cutoff (-gr)
ΔCT norm Normalized ΔCT = [CT(mutant allele assay negative control) – CT(gene reference assay)] - Calibration ΔCT - (3 × the standard
deviation or 2 CT, whichever is greater).

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
NOTICE TO PURCHASER: PLEASE REFER TO THE TAQMAN® MUTATION DETECTION ASSAYS PROTOCOL PRODUCT INSERT AND PROTOCOL FOR LIMITED LABEL LICENSE OR
DISCLAIMER INFORMATION.
LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT
LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE
TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL,
INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.
© 2012 Life Technologies Corporation. All rights reserved. of Life Technologies Corporation or their respective owners. TaqMan is a registered
trademark of Roche Molecular Systems, Inc.

Headquarters
5791 Van Allen Way | Carlsbad, CA 92008 USA | Phone +1 760 603 7200 | Toll Free in USA 800 955 6288
For support visit www.lifetechnologies.com/support
www.lifetechnologies.com