HID Real‑Time PCR Analysis Software

Version 1.3

for use with:

7500 Real-Time PCR Instrument
QuantStudio™ 5 Real-Time PCR Instrument (with 0.2-mL 96-Well Block)
Quantifiler™ DNA Quantification Kit
Publication Number MAN0009819
Revision E.0

For Forensic or Paternity Use Only.

 Manufacturer: Thermo Fisher Scientific | 7 Kingsland Grange | Warrington, Cheshire WA1 4SR | United Kingdom

The information in this guide is subject to change without notice.

DISCLAIMER: TO THE EXTENT ALLOWED BY LAW, THERMO FISHER SCIENTIFIC INC. AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL,
INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT,
INCLUDING YOUR USE OF IT.

Revision history: Pub. No. MAN0009819

Revision Date Description
E.0 27 August 2018 Updated branding and trademarks, no technical changes.
™ ™
D.0 8 March 2017 Add support for the Applied Biosystems QuantStudio 5 Real-Time PCR
System with 96-well (0.2-mL) sample block.
Add Virtual Standard Curve function.
™ ™
C.0 14 August 2015 Correct quencher listed for Quantifiler Duo and Quantifiler Male or
Human kits.
Add reference to evaluating the quality indices determined by the
HID Real‑Time PCR Analysis Software to determine if highly degraded
™
samples can be better analyzed with the Ion Personal Genome Machine
(PGM ) System. See the Quantifiler HP and Trio DNA Quantification Kits
™ ™

User Guide.
B.0 March 2014 Added Chapter 9, “HID Real-Time PCR Analysis Software Validation”.
™
A.0 January 2014 New document for version 1.2 features (support for Quantifiler Trio and
HP DNA Quantification Kits; Degradation Index). Incorporates all
information from the HID Real-Time PCR Analysis Software v1.1 User
Guide (Pub. no. 4455443)

Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept
the terms and conditions of all applicable Limited Use Label Licenses.
TRADEMARKS: All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. TaqMan is a registered
trademark of Roche Molecular Systems, Inc, TaqMan used under permission and license. Microsoft, Windows, PowerPoint, and Excel are registered
trademarks of Microsoft Corporation. Adobe, Acrobat, and Reader are registered trademarks of Adobe Systems, Inc. Pentium and Intel are registered
trademarks and Core is a trademark of Intel Corporation.
©2018 Thermo Fisher Scientific Inc. All rights reserved.
 Contents

About This Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

CHAPTER 1 Get Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Software overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Features in v1.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Install or upgrade to v1.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Calibrate the 7500 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Calibrate the QuantStudio™ 5 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

CHAPTER 2 Customize the Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Modify a default experiment template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Create an experiment template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Link your template to a Home screen button . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
Set display defaults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

CHAPTER 3 Select the Experiment and

Set Up a Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
HID Real-Time PCR Analysis Software workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Start the software and select an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
Navigate the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
Specify experiment properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
Define samples and view targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Assign the targets, samples, and standards to wells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Save plate layout as *.eds or template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Link your template to a Home screen button . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

CHAPTER 4 Run the Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

View the run method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
Set notifications (7500 System Only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
Start or stop the run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Monitor the run (7500 System Only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Save the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 3

Contents

CHAPTER 5 Select Analysis Settings and Thresholds . . . . . . . . . . . . . . . . . . . . . 43

Open analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
View/Edit CT settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Enter HID settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
Enter Flag settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Add a virtual standard curve to the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

CHAPTER 6 Enhance Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

View the analysis results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Interpret QC flag information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
Omit wells from analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Omit targets in an experiment well . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Examine the Degradation Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Change the appearance of, print, and save plots . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

CHAPTER 7 Export and Report Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Export data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Print a report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

CHAPTER 8 Generate Dilution and Reaction Worksheets for STR Setup . . . . 63

Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Add kits to an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Select unknown samples for amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
Edit dilution settings for individual samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
View the dilution scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Export dilution and reaction worksheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Save new STR Kit information from an experiment into STR Kit Library . . . . . . . . . . . . . . . . 66

CHAPTER 9 HID Real-Time PCR Analysis Software Validation . . . . . . . . . . . . . 67

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Experiments and results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

APPENDIX A Configure STR Library and Default Dilution Settings . . . . . . . . . . 73

Configure the STR Kit Library . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

4 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Contents

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

How to use your documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Obtaining related documentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Obtaining information from the Help system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Customer and technical support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 5

Contents

6 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 About This Guide

Purpose
The 7500 Real-Time PCR System, or the QuantStudio™ 5 Real-Time PCR Instrument,
and the HID Real-Time PCR Analysis Software detects and quantifies human and/or
male DNA in samples. This guide is intended to help you quickly learn how to use the
HID Real-Time PCR Analysis Software to perform analysis of samples prepared with
the:
• Quantifiler™ HP DNA Quantification Kit
• Quantifiler™ Trio DNA Quantification Kit
• Quantifiler™ Human DNA Quantification Kit
• Quantifiler™ Duo DNA Quantification Kit
• Quantifiler™ Y Human Male DNA Quantification Kit
This guide assumes that:
• You are familiar with the Microsoft® Windows® operating system, the Internet,
and Internet browsers.
• You know how to handle DNA samples and prepare them for PCR.
Use this guide after your plate is prepared and loaded in the 7500 Real-Time PCR
System or QuantStudio™ 5 Real-Time PCR Instrument (with 0.2-mL 96-Well Sample
Block).

For instructions on preparing a plate, refer to the user guide for the Quantifiler™
Kit you are using.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 7

About This Guide
Purpose

8 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 CHAPTER 1

Get Started
1
This chapter covers:
n Software overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
n Features in v1.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
n Install or upgrade to v1.3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
n Calibrate the 7500 Instrument. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
n Calibrate the QuantStudio™ 5 Instrument . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Software overview
HID Real-Time PCR Analysis Software is designed specifically to assist human
identification laboratories performing DNA quantitation, by simplifying assay setup
and streamlining data review and dilution and reaction setup for downstream STR
analysis. For example, the software automatically selects the appropriate Quantifiler™
Kit target, reporter, quencher, and thermal profile. After a run, the HID Real-Time PCR
Analysis Software provides an analysis of each well. The software exports:
• All results
• STR kit setup instructions
• Sample dilutions calculations
HID Real-Time PCR Analysis Software is for use with the 7500 Real-Time PCR
Instrument and the QuantStudio™ 5 Real-Time PCR Instrument (with 0.2-mL 96-Well
Sample Block).

Applicable HID kits

You can use the HID Real-Time PCR Analysis Software with the following kits:
• Quantifiler™ HP DNA Quantification Kit
• Quantifiler™ Trio DNA Quantification Kit
• Quantifiler™ Human DNA Quantification Kit
• Quantifiler™ Duo DNA Quantification Kit
• Quantifiler™ Human Male DNA Quantification Kit

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 9

 Chapter 1 Get Started
1 Features in v1.3

Custom experiment option

IMPORTANT! The custom assay option is supported for the 7500 system only.

You can also use the HID Real-Time PCR Analysis Software for more complex
experiments by selecting the Custom Assay option on the Home screen. If you use the
Custom Assay option, refer to the Applied Biosystems 7500/7500 Fast Real-Time PCR
System Getting Started Guide for Standard Curve Experiments for instructions.

Features in v1.3
HID Real-Time PCR Analysis Software v1.3 includes all of the v1.2 functionality and
includes the following new features:
• Virtual Standard Curve support for Quantifiler™ HP, Trio, Duo, and Human DNA
Quantification Kits.
• Support for the QuantStudio™ 5 Real-Time PCR Instrument with 0.2-mL 96-Well
Sample Block.

Install or upgrade to v1.3

Refer to the 7500/7500 Fast Real-Time PCR System Site Preparation Guide
(Pub. no. 4412843) for system layout, electrical, power, safety, and other site
requirements.
Follow the appropriate installation procedure:

Situation See…

Install the software with a new instrument page 11

Upgrade from HID Real-Time PCR Analysis Software v1.1 to v1.3 page 12
Upgrade from HID Real-Time PCR Analysis Software v1.2 to v1.3 page 12

10 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 1 Get Started
Install or upgrade to v1.3 1
Computer and instrument requirements

Component Requirements

Computer • Processor (minimum: 2.9 GHz):

– (Recommended) Intel™ Core™ i7 CPU, 2.9 GHz
– Intel™ Core™ i5 Quad Core CPU, 2.9 GHz
• 16 GB RAM1
• One hard drive with at least 10 GB available
• 20/48X IDE CD-ROM drive
• USB v1.2
• Ethernet network interface adapter (10BASE-T)2§
• Microsoft™ Windows™ 7 64-bit/32-bit, Service Pack 1 or later
Software • Microsoft™ PowerPoint™ software (for direct export of PowerPoint slides)
• Microsoft™ Excel™ software (for direct export of data to spreadsheet)
IMPORTANT! Do not run antivirus applications while HID Real-Time PCR Analysis Software v1.3 is
running.
Monitor • 1280 × 1024 pixel resolution for full screen display3
• 16-inch
• True Color (32 bit)
• UL listed
Instrument 7500 Real-Time PCR System
Instrument firmware G2.10 (installed on all instruments
purchased after 2008)
If your instrument was purchased before 2008, check the version of firmware installed by going to x:/
Applied Biosystems/7500 system/firmware. The example below shows firmware G2.09. Contact Thermo
Fisher Scientific if your firmware version is not G2.10.
QuantStudio™ 5 Real-Time PCR Instrument
Instrument firmware 1.3.1 or later
To check the firmware version of your instrument, from the Home screen, touch SettingsAbout
InstrumentAbout Instrument.
1 The software may experience communication errors if run on computers with less than 1 GB.
2 Required only if you plan to connect the computer to a local area network (LAN).
3 If screen resolution is not set to 1280 X 1024, the Analysis Summary screen may not be properly displayed.

Install with a new instrument

If the HID Real-Time PCR Analysis Software is installed with a new 7500 Real-Time
PCR System or QuantStudio™ 5 Real-Time PCR Instrument, both the instrument and
the software must be installed by an Thermo Fisher Scientific technical representative.
If you have the instrument but no 7500 Software installed, you can install v1.3 directly.
Insert the HID Real-Time PCR Analysis Software CD and follow the Installation
Wizard instructions.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 11

 Chapter 1 Get Started
1 Install or upgrade to v1.3

Upgrade from v1.1 to v1.3

IMPORTANT! You must have Administrator privileges to perform the upgrade.

1. Uninstall the HID Real-Time PCR Analysis Software v1.1 from your computer.
IMPORTANT! You must uninstall HID Real-Time PCR Analysis Software v1.1
before you follow this procedure. If v1.1 is present, the installation will fail.

2. Insert the HID Real-Time PCR Analysis Software v1.3 CD.

3. Follow the Installation Wizard instructions.

4. Make sure that all calibrations are up-to-date. See “Calibration procedure” on
page 14 for instrument calibration requirements.

Upgrade from v1.2 to v1.3

IMPORTANT! Do not uninstall v1.2 before performing the upgrade. If v1.2 is not
present, the upgrade will fail.

IMPORTANT! You must have Administrator privileges to perform this upgrade.

1. Insert the HID Real-Time PCR Analysis Software v1.3 CD.

The v1.3 upgrade installer automatically backs up the v1.2 calibration,
experiments, and log files, then uninstalls v1.2. After the v1.3 software installation
is complete, you can restore the backed-up files to the appropriate folders as
described in step 4.

2. Follow the Installation Wizard instructions.

IMPORTANT! If installing on a computer connected to the instrument, enter the
same instrument serial number that was entered when v1.2 was installed.

3. Enter the Upgrade Registration Code provided with the installation CD. Do not
enter any spaces between the numbers in the registration code.

4. (If installing on a computer connected to the instrument) After installation

sucessfully completes, copy the calibration and experiments files from the back-
up folders to the appropriate folders:

Calibration files From \<install location>\7500\backup\calibration\<date stamp>\eclipse\plugins\

com.apldbio.sds.instrument.sds7500_1.0.0\config\
To \<install location>\7500\eclipse\plugins\
com.apldbio.sds.instrument.sds7500_1.0.0\config\
Experiments files From \<install location>\7500\backup\experiments\<date stamp>
To \<install location>\7500\experiments
Note: During installation, the v1.2 log files are backed up to \<install location>\
7500\backup\config\logs\<date stamp>. The files do not need to be reinstalled.

5. Make sure that all calibrations are up-to-date. See “Calibration procedure” on
page 14 for instrument calibration requirements.

12 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 1 Get Started
Install or upgrade to v1.3 1
Replace SDS Software v1.2.3
1. Review “Computer and instrument requirements” on page 11 and ensure that
your system meets the requirements.

2. Archive all experiment and calibration data.

3. Uninstall the SDS Software v1.2.3.

IMPORTANT! If the SDS Software v1.2.3 is present, the installation will not run.

4. Insert the HID Real-Time PCR Analysis Software v1.3 CD.

5. Enter the Full Installation Registration Code provided with the installation CD.

6. Follow instructions of the installation wizard.

7. Make sure that all calibrations are up-to-date. See “Calibration procedure” on
page 14 for instrument calibration requirements.

7500 Real-Time PCR Instruments purchased before February 2008

Tower and laptop computers of 7500 Real-Time PCR Instruments purchased before
February 2008 require a memory upgrade before the computers can install the HID
Real-Time PCR Analysis Software. Refer to the Applied Biosystems 7500/7500 Fast Real-
Time PCR Systems User Bulletin Memory Upgrade Requirements for 7500 Software v2.0
(Pub. no. 4379705) for information.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 13

 Chapter 1 Get Started
1 Calibrate the 7500 Instrument

Calibrate the 7500 Instrument

IMPORTANT! The following procedure is for the 7500 Real-Time PCR Instrument only.
If you are using a QuantStudio™ 5 Instrument, see “Calibrate the QuantStudio™ 5
Instrument” on page 16.

If you... Perform...
Installed HID Real-Time PCR Perform all calibrations and run the RNase P plate
Analysis Software v1.3 with a
new instrument
Upgraded from HID Real-Time After restoring v1.2 calibration files (see “Upgrade from
PCR Analysis Software v1.2 v1.2 to v1.3” on page 12), perform Custom Dye calibration
to calibrate ABY™, JUN™ and Mustang Purple™ (MP) dyes.
Replaced SDS Software v1.2.3 Perform all calibrations and run the RNase P plate

Required materials
Table 1 lists the materials that are required to required to calibrate the instrument.

Table 1 User-supplied materials

If you... Material Cat. no.

Replaced SDS 7500 Real Time PCR Systems Spectral Calibration Kit I 4349180
Software v1.2.3
TaqMan™ RNase P Instrument Verification Plate 4350584
Upgraded from HID 96-Well Spectral Calibration Plate with ABY™ Dye 4461591
v1.2
96-Well Spectral Calibration Plate with JUN™ Dye 4461593
or
96-Well Spectral Calibration Plate with 4461599
Replaced SDS v.1.2.3 Mustang Purple™ Dye

Calibration procedure
The following is an outline of the calibration procedure. See the Applied Biosystems™
7500/7500 Fast Real-Time PCR Systems System Maintenance Guide (Pub. no. 4387777) for
complete instructions.
Perform:
• Regions of Interest (ROI) calibration
• Background Calibration
• Optical Calibration
• Dye Calibration:
– Perform Dye Calibration of the new ABY™, JUN™ and Mustang Purple™
(MP) dyes. Follow the custom dye procedure.
– Perform Dye Calibration of all system dyes for new instrument installations,
or if replacing SDS v.1.2.3
– Use 60°C as the default temperature for all dye calibration
• RNase P Instrument Verification Plate run

14 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 1 Get Started
Calibrate the 7500 Instrument 1
New dye spectra
Figure 1 through Figure 3 show the calibration spectra for ABY™, JUN™ and
Mustang Purple™ (MP) dyes.

Figure 1 ABY™ dye spectra

Figure 2 JUN™ dye spectra

Figure 3 Mustang Purple™ (MP) dye spectra

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 15

 Chapter 1 Get Started
1 Calibrate the QuantStudio™ 5 Instrument

Calibrate the QuantStudio™ 5 Instrument

IMPORTANT! The following procedure is for the QuantStudio™ 5 Real-Time PCR
Instrument only. If you are using a 7500 System, see “Calibrate the 7500 Instrument”
on page 14.

The QuantStudio™ 5 Real-Time PCR Instrument is calibrated during manufacturing;

however, you must recalibrate the instrument for the dyes that are used for HID
analysis before use. If you installed HID Real-Time PCR Analysis Software v1.3 with
a new instrument, perform Custom Dye calibrations for the ABY™ and JUN™ HID
dyes.

Required materials
Table 2 lists the materials that are required to calibrate the instrument.

Table 2 User-supplied materials

Material Cat. no.

96-Well Spectral Calibration Plate with ABY™ Dye 4461591
96-Well Spectral Calibration Plate with JUN™ Dye 4461593
TaqMan™ RNase P Instrument Verification Plate 4432382

Calibration procedure
The following is an outline of the calibration procedure. See the QuantStudio™ 3 and 5
Real-Time PCR Systems Installation, Use, and Maintenance Guide (Pub. no. MAN0010407)
for complete instructions.
Perform:
• Dye Calibration:
– Perform Dye Calibration of the new ABY™ and JUN™ dyes. Follow
the custom dye procedure.
– Use 60°C as the default temperature for all dye calibrations.
IMPORTANT! You must calibrate the ABY™ Dye as ABY-HID and the JUN™ Dye
as JUN-HID. Calibrating either dye without the “-HID” suffix (as ABY and JUN)
overwrites the existing calibrations for the factory-calibrated system dyes. Doing
so potentially creates confusion if the instrument is ever calibrated using the
QuantStudio™ 3 and 5 Calibration Kit, which lacks the HID versions of the dyes.
• RNase P Instrument Verification Plate run

16 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 1 Get Started
Calibrate the QuantStudio™ 5 Instrument 1
New dye spectra
Figure 4 through Figure 6 show the calibration spectra for ABY™, JUN™, and
Mustang Purple™ (MP) dyes.

Figure 4 ABY™ dye spectra

Figure 5 JUN™ dye spectra

Figure 6 Mustang Purple™ (MP) dye spectra

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 17

 Chapter 1 Get Started
1 Calibrate the QuantStudio™ 5 Instrument

18 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 CHAPTER 2

Customize the Software

2
This chapter covers:
n Modify a default experiment template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
n Create an experiment template. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
n Link your template to a Home screen button. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
n Set display defaults . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Modify a default experiment template

You can make changes to the experiment templates provided with the software after
making a backup copy of the original templates.

Save a copy of the original template

Before you modify a template, save a copy of the original template:

1. Navigate to: C:\Applied Biosystems\7500\config\templates

2. Select EditCopy to copy the templates folder.

3. Navigate to a safe location on your computer.

4. Select EditPaste to insert a copy of the templates folder in the location you
selected.

Modify the original template

1. Click the button on the Home screen for the experiment type of interest.

2. Modify the template as needed, including:

• Moving standards and NTCs to different wells
• Adding samples and/or extraction blanks
• Setting plate layout
• Setting the display defaults for the Amplification plot, plate view, and well
table
• Modifying analysis settings (HID, CT, and Flags)

3. Click the down arrow next to Save in the toolbar, then in the drop-down list, then
select Save as, then select the name of the original template.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 19

 Chapter 2 Customize the Software
2 Create an experiment template

Create an experiment template

1. Set up an experiment with the desired settings, including:
• Moving standards and NTCs to different wells
• Adding samples and/or extraction blanks
• Setting plate layout
• Setting the display defaults for the Amplification plot, plate view, and well
table
• Modifying analysis settings (HID, CT, and Flags)

2. Click the down arrow next to Save in the toolbar, then select Save as Template.
To use your template instead of a default template, click Open at the top of the Home
screen, then select your template instead of clicking a button for an experiment type.

Link your template to a Home screen button

You can link your template to the any of the Quantifiler™ assay icons on the Home
screen for:
The software will automatically use the template as the default experiment when you
click the corresponding button. You will still be able to use a different template by
opening a different experiment.

1. Before you link your template file to a button on the Home screen, save a copy of
the original template:
a. Navigate to: C:\Applied Biosystems\7500\config\templates
b. Select EditCopy to copy the templates folder.
c. Navigate to a safe location on your computer.
d. Select EditPaste to insert a copy of the templates folder in the location you
selected.

2. Link your template to a button on the Home screen:

a. In the toolbar, from the file that you want to link, click the down arrow next
to Save.
b. In the drop-down menu, select Save as Template.
c. Navigate to: C:\Applied Biosystems\7500\config\templates

20 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 2 Customize the Software
Set display defaults 2
d. Select the file corresponding to the assay button that you want to replace.
IMPORTANT! Files that contain the "QS5" suffix are templates used by the
QuantStudio™ 5 Instrument. For example, the "QuantifilerTrio.edt" is the
template file for the Quantifiler™ Trio Kit used by 7500 instruments, whereas
"QuantifilerTrioQS5.edt" is the file used by QuantStudio™ 5 Instruments.

IMPORTANT! Be sure to give the file exactly the same name as the file
corresponding to the button that you want to replace.
e. Click Save.

Set display defaults

Select information to display in the Plate View
1. Click Show in Wells to open the drop-down list.

2. Click the checkbox next to an item of data to select ( ) the item for display or to
deselect it ( ).

3. Click Set as Default (this button is dimmed before you change a setting, or if you
are logged in as Guest).

Specify the information to display in the Well Table

1. Click Show in Table to open the drop-down list.

2. Click the checkbox next to an item of data to select ( ) the item for display or to
deselect it ( ).

3. Click Set as Default (this button is dimmed before you change a setting, or if you
are logged in as Guest).

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 21

 Chapter 2 Customize the Software
2 Set display defaults

Customize the amplification plot

1. Make changes as described in “Change the appearance of, print, and save plots”
on page 57.

2. Click the checkbox next to an item of data to select ( ) the item for display or to
deselect it ( ).

3. Click Save Current Settings as Default (this button is dimmed before you change
a setting, or if you are logged in as Guest).

22 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 CHAPTER 3

Select the Experiment and

3 Set Up a Plate

This chapter covers:

n HID Real-Time PCR Analysis Software workflow . . . . . . . . . . . . . . . . . . . . . . . . . 24
n Start the software and select an experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
n Navigate the software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
n Specify experiment properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
n Define samples and view targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
n Assign the targets, samples, and standards to wells . . . . . . . . . . . . . . . . . . . . . . . . 30
n Save plate layout as *.eds or template . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
n Link your template to a Home screen button. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
This chapter assumes that you have prepared a plate according to the instructions
in the user guide for the Quantifiler™ Kit you are using.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 23

 Chapter 3 Select the Experiment and Set Up a Plate
3 HID Real-Time PCR Analysis Software workflow

HID Real-Time PCR Analysis Software workflow

Set up a Quantifiler™ chemistry kit plate and load in the

instrument.

Select the experiment and set up a plate:

1. Start the software.
2. Start a new experiment.
3. Specify experiment properties.
4. Define samples and assign targets, samples, and

HID Real-Time PCR Analysis Software

standards to wells.
5. Save the experiment.

Run the plate.

Select analysis settings and thresholds:

• HID settings (includes HID flags)
• Flag settings

Review results:
1. View analysis summary.
2. View quantitation results.

Export and print results.

Generate dilution and reaction worksheets for STR set

up:
1. Configure STR Library and default dilution settings.
2. Add kits to an experiment.
3. Select the unknown samples for amplification.
4. Edit dilution settings for individual samples as
needed.
5. Export dilution and reaction worksheets.

Perform PCR amplification.

24 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 3 Select the Experiment and Set Up a Plate
Start the software and select an experiment 3
Start the software and select an experiment
1. On your desktop, double-click or select StartAll ProgramsApplied
BiosystemsHID Real-Time PCR Analysis SoftwareHID Real-Time PCR
Analysis Software. The Login Screen should open within 1 minute.

2. In the User Name field, enter your user name or select it from the drop-down list.
You can log in as a guest, but only users logged in with a user name can:
• Edit the names of folders for experiment information import, information
export, or data.
• Enable or disable the requirement to enter a user name to start the software.
• Set a plate layout as the default layout (See “Link your template to a Home
screen button” on page 35).
• Configure how data is displayed (see Chapter 2, “Customize the Software”).

3. Click OK to open the Home screen with icons for HID and Custom Assays as
shown.

Quantifiler™ assays Custom assays

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 25

 Chapter 3 Select the Experiment and Set Up a Plate
3 Navigate the software

4. Choose an HID experiment:

• Click one of the HID template icons:
– Quantifiler™ HP
– Quantifiler™ Trio
– Quantifiler™ Duo
– Quantifiler™ Male
– Quantifiler™ Human
– Quantifiler™ Male & Human (hybrid plate)
or
• In the toolbar, click the down arrow next to New Experiment to open the
drop-down list and select the appropriate experiment.

For custom experiments

IMPORTANT! The custom experiments feature is supported for the 7500 system only.

To perform a non-HID experiment, or a modified experiment, click:

• Custom Assay on the right side of the Home screen.
or
• Assays in the toolbar, then select Custom Assays in the drop-down list.
For information on running custom experiments, refer to the 7500/7500 Fast Real-Time
PCR System Getting Started Guide for Standard Curve Experiments.

Navigate the software

Each HID Real-Time PCR Analysis Software experiment screen displays

instructions for a step in the experiment. Use the Experiment Menu at the
left of any screen to navigate the software.
Click >> (Expand) to expand the Experiment Menu.
Click << (Collapse) to collapse the Experiment Menu.
Click Setup, Run, or Analysis, to display screens used in the
corresponding process.
You can access HID Real-Time PCR Analysis Software screens in any
sequence.
To return to the Home screen at any time, click (Home) at the bottom
left of any screen.

26 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 3 Select the Experiment and Set Up a Plate
Specify experiment properties 3
Specify experiment properties

1. In the Experiment Menu, select SetupExperiment Properties.

2. In the “How do you want to identify this experiment?” section, enter the name of
the plate or experiment information in the Experiment Name field. Entries in the
other fields are optional.

Note: The name you enter in the Experiment Name field appears on the data report
and on *.xls spreadsheets of data that you export. If you do not enter a name,
“Untitled” appears on the report and in the exported spreadsheet.

The following parameters are automatically set:

• Experiment Name: Untitled
• Instrument:
– 7500 (96 wells)
– QuantStudio 5 (96 wells)
• Experiment Type: Quantitation-HID Standard Curve
• Reagents: TaqMan™ Reagents
• Ramp Speed: Standard (~1 hour to complete a run for Quantifiler™ HP
and Quantifiler™ Trio kits, and ~2 hours for all other Quantifiler™ kits)

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 27

 Chapter 3 Select the Experiment and Set Up a Plate
3 Define samples and view targets

Define samples and view targets

Note: Targets are automatically listed and named. Standards dilutions and an NTC
sample are listed by default for each Quantifiler™ Kit. For information about the
standard included in the Quantifiler™ Kit, refer to your Quantifiler™ Kit user guide
(see “How to use your documentation” on page 77).

Define samples
1. In the Experiment Menu, click SetupPlate Setup. Select the Define Targets and
Samples tab.

2. In the Define Samples area on the right side of the pane, specify sample names.

• To define a new sample:

– Click Add New Sample. A new line appears in the Sample Name field,
or
– In the toolbar, click ToolsSample Library to open the Sample Library
screen, then click New.
The default name for the new sample is Sample X (where X=1 or the highest
listed Sample # + 1). You can enter a new name for the sample. To save the
name of the sample for future experiments, click OK.
• To use a sample from your sample library:
a. In the Define Samples pane, select Add Saved Sample.
b. Select the sample(s) to use, then click Add Selected Sample(s).
Note: You can also add a sample to a single well in the Plate Setup screen. See
“Assign a new sample to a well” on page 32.

28 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 3 Select the Experiment and Set Up a Plate
Define samples and view targets 3
3. Select the sample type: Standard, NTC, or Unknown.
Unknown is the default sample type for new samples.
When you assign the sample type, the software automatically
assigns the appropriate task to each target.

4. Repeat steps 2 and 3 for each sample.

IMPORTANT! List each sample individually. For replicates (identical samples), add
the sample name only once. To assign a replicate to a well in the plate, in step 4 on
page 31, select the well, then select the checkbox next to the sample name.

View targets
1. In the Experiment Menu, select SetupPlate Setup.

2. Select the Define Targets and Samples tab.

3. In the Defined Targets area on the left side of the pane, view the targets list to
verify that you selected the correct experiment in step 4 on page 26.

Kit Reporter dyes Quencher

Quantifiler™ Trio Small autosomal: VIC™ dye NFQ-MGB

Male (Y): FAM™ dye NFQ-MGB
Large autosomal: ABY™ dye QSY7
IPC: JUN™ dye QSY7
Quantifiler™ HP Small autosomal: VIC™ dye NFQ-MGB
Large autosomal: ABY™ dye QSY7
IPC: JUN™ dye QSY7
Quantifiler™ Duo Human: VIC™ dye NFQ-MGB
Male: FAM™ dye NFQ-MGB
IPC: NED™ dye NFQ-MGB
Quantifiler™ Male Human: FAM™ dye NFQ-MGB
or Human
Male: FAM™ dye NFQ-MGB
IPC: VIC™ dye NFQ-MGB
Quantifiler™ Human Human: FAM™ dye NFQ-MGB
IPC: VIC™ dye NFQ-MGB
Quantifiler™ Male Male: FAM™ dye NFQ-MGB
IPC: VIC™ dye NFQ-MGB

Change color To change the color that represents a target in the data analysis:
designation
1. Click (down arrow) in the Color column.

2. Select a color in the drop-down list.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 29

 Chapter 3 Select the Experiment and Set Up a Plate
3 Assign the targets, samples, and standards to wells

Assign the targets, samples, and standards to wells

Go to the Assign Targets and Samples tab.
• In the Define Targets and Samples tab, click Assign
Targets and Samples beneath the Define Samples area.
or
• In the Experiment Menu, select SetupPlate Setup, then select the Assign
Targets and Samples tab.

Assign Using Plate Layout

Assign samples, To assign samples, standards, and NTCs using the View Plate Layout tab:
standards, and NTCs
to wells 1. Select the View Plate Layout tab in the pane on the right of the screen.
To select wells with specific characteristics:
a. Click the left Select Wells With button above the layout diagram.
b. Select Sample, Target, or Task in the drop-down list.
c. Click the right Select Wells With button.
d. Select a specific sample, target, or task.

30 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 3 Select the Experiment and Set Up a Plate
Assign the targets, samples, and standards to wells 3
2. Specify the information to display in the wells:
a. Click Show in Wells to open the drop-down list. Items that are marked with
a check ( ) are selected for display.
b. Click an item to select or deselect it for display.

3. (Optional) To save your selections as default settings, click Set as Default at the
top right of the View Plate Layout toolbar.

4. Assign standards, NTCs, and unknown samples to well(s).

a. To select:
• Well – Click the well
• Row of wells – Click a letter on the side of the layout
• Column of wells – Click a number at the top of a column
• More than one well, row, or column – Drag the pointer over the wells,
letters, or columns to select
b. In the Assign Sample(s) to the Selected Wells section to the left of the plate
layout, select the check box in the Assign column corresponding to the
unknown, standard, or NTC sample in the well(s). The target for each
sample is set by default.

Note: <Sample 1> is automatically assigned to all wells that are not assigned
as standard(s) or NTC(s).

5. (Optional) To change the quantity of standards, enter the quantity in ng/µL in the
Quantity field in the Assign Targets to the Selected Wells area. The quantity of
standard samples is set by default.

6. Repeat steps 4 and 5 until you assign samples, standards, and NTCs to all wells
that you use in the experiment. You can delete empty wells after data analysis.
Note: If you delete the samples/standards/NTCs in a well and then restore them,
you must reenter the well information.
The task for each target/sample combination is set automatically.

7. Clear all wells that do not contain samples or targets:

a. Select the well(s) to clear.
b. Right-click the well(s).
c. Select Clear from the drop-down list.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 31

 Chapter 3 Select the Experiment and Set Up a Plate
3 Assign the targets, samples, and standards to wells

Assign a new
sample to a well

To add a new sample to a well:

1. Double-click the well to open the Add New Sample dialog box.

2. Click Add New Sample.

3. Target and task are set by default according to sample type. To change the sample
type, click the down arrow in the Sample column header and select the
appropriate sample type from the drop-down list.

4. To change the sample quantity setting for standard samples, perform step 5 on
page 31.

Move samples, 1. Select the wells for the samples, standards or NTCs you want to move.
standards, and NTCs
2. Deselect ( ) the items in the Assign Sample(s) to the Selected Wells pane, or
right-click the wells and select Clear.

3. One at a time, select the new wells for an item you are moving, then select ( ) the
items in the Assign Sample(s) to the Selected Wells pane.

32 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 3 Select the Experiment and Set Up a Plate
Assign the targets, samples, and standards to wells 3
Assign Using Well Table

To assign samples, standards, and NTCs using the View Well Table tab:

1. Select the View Well Table tab.

Each row in the table represents one well. To group the rows by a characteristic,
click the column header. For example, click Task to group rows by task.
To select wells with specific characteristics:
a. Click the left Select Wells With button above the layout diagram.
b. Select Sample, Target, or Task in the drop-down list.
c. Click the right Select Wells With button.
d. Select a specific sample, target, or task.

2. Specify the information to display in the table:

a. Click Show in table to open the drop-down list. Items that are checked in the
check box ( ) are selected for display.
b. Click an item to select or deselect it for display.

3. (Optional) To save your selections as default settings, click Set as Default at the
top right of the View Plate Layout toolbar.

4. Assign samples, standards, and NTCs to well(s):

a. Select the well(s). To select:
• Well – Click under one of the column headings in the row next to the
well location (for example, to select well A6, click in row A6 under
Sample).
• More than one well – Drag the pointer over the wells that you want to
select, or Ctrl+Click the wells that you want to select.
b. In the Assign Sample(s) to the Selected Wells section, select the check box in
the Assign column corresponding to the unknown, standards, or NTC
sample in the well(s). The target for each sample is set by default.

Note: <Sample 1> is automatically assigned to all wells that are not assigned
as standard(s) or NTC(s).

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 33

 Chapter 3 Select the Experiment and Set Up a Plate
3 Save plate layout as *.eds or template

5. (Optional) To change the quantity of standards, enter the quantity (in ng/µL) in
the Quantity field in the Assign Targets to the Selected Wells area. The quantity of
samples is set by default.

6. Repeat steps 1 and 5 until you assign samples, standards, and NTCs to all wells
that you use in the experiment. You can delete empty wells after data analysis.
Note: If you delete the samples, standards, or NTCs in a well and then restore
them, you must reenter the well information.
The task for each target/sample combination is set automatically.

7. Clear all wells not assigned:

a. Click the left Select Wells With button at the top of the table.
b. Select Sample from the drop-down list.
c. In the well table, select the sample name(s) of the well(s) to clear.
d. In the Assign samples to the selected wells area, deselect the checkbox in the
Assign column beside the sample name.

Save plate layout as *.eds or template

IMPORTANT! Do not save the experiment to the network folder until the plate run is
completed.

1. To save your plate layout, in the toolbar, click the down arrow next to Save, then
in the drop-down list, select:
• Save – to save the plate layout as an Experiment Document Single (*.eds)
file
• Save as – to save the plate layout as a *.eds file with a different name
or
• Save as Template – to save the experiment file as a template for future
experiments

2. If you want to save the file with a different name, enter the new name in the File
Name field.

3. Click Save.

4. Before you start the run, verify that the plate is loaded in the instrument, as
described in the user guide for the Quantifiler™ Kit you are using.

34 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 3 Select the Experiment and Set Up a Plate
Link your template to a Home screen button 3
Link your template to a Home screen button
You can link your template to the any of the Quantifiler™ assay icons on the Home
screen for:
The software will automatically use the template as the default experiment when you
click the corresponding button. You will still be able to use a different template by
opening a different experiment.

1. Before you link your template file to a button on the Home screen, save a copy of
the original template:
a. Navigate to: C:\Applied Biosystems\7500\config\templates
b. Select EditCopy to copy the templates folder.
c. Navigate to a safe location on your computer.
d. Select EditPaste to insert a copy of the templates folder in the location you
select.

2. Link your template to a button on the Home screen:

a. In the toolbar, from the file that you want to link, click the down arrow next
to Save.
b. In the drop-down menu, then select Save as Template.
c. Navigate to: C:\Applied Biosystems\7500\config\templates
d. Select the file corresponding to the assay button that you want to replace.
IMPORTANT! Files that contain the "QS5" suffix are templates used by the
QuantStudio™ 5 Instrument. For example, the "QuantifilerTrio.edt" is the
template file for the Quantifiler™ Trio Kit used by 7500 instruments, whereas
"QuantifilerTrioQS5.edt" is the file used by QuantStudio™ 5 Instruments.

IMPORTANT! Be sure to give the file exactly the same name as the file
corresponding to the button that you want to replace.
e. Click Save.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 35

 Chapter 3 Select the Experiment and Set Up a Plate
3 Link your template to a Home screen button

36 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 CHAPTER 4

Run the Plate

4
This chapter covers:
n View the run method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
n Set notifications (7500 System Only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
n Start or stop the run . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
n Monitor the run (7500 System Only) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
n Save the results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 37

 Chapter 4 Run the Plate
4 View the run method

View the run method

1. In the Experiment Menu, select SetupRun Method to open the Run Method
screen.

2. Select the Graphical View tab to open the thermal profile for the assay.
Note: The Graphical View tab displays the Run Method ramp rate as a
percentage when using a 7500 System and in degrees Celsius (°C) when using a
QuantStudio™ 5 Instrument.

Kit 7500 System QuantStudio™ 5 System

Thermal
profile for
Duo Kit

Thermal
profile for
Male Kit,
Human Kit,
and
Human and
Male Kit

3. Verify that the value in the reaction volume field is:

• 25 µL for Quantifiler™ Human, Human Male, and Duo Kits
• 20 µL for Quantifiler™ HP and Trio Kits
For more information on run parameters, refer to the user guide for
the Quantifiler™ Kit you are using.

38 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 4 Run the Plate
Set notifications (7500 System Only) 4
Set notifications (7500 System Only)
IMPORTANT! The following procedure is for the 7500 Real-Time PCR Instrument only
and not available for the QuantStudio™ 5 Real-Time PCR Instrument.

You can set the software to send e-mail notification of selected events to e-mail
addresses that you specify.

1. In the Experiment Menu, select RunNotification

Settings to open the screen.

2. To send notifications:
• In the Run Status area, select the Enable Notifications check box.
or
• In the Notifications Settings area, select Yes for Enable Notifications. If you
do not want the system to send notifications, select No.

IMPORTANT! Notifications cannot be sent unless the computer that performs the
run is on an e-mail network.

3. For “Select the events to generate notifications,” select the check boxes for events
that you want to generate e-mails. You can select:
• Instrument Error – Notifies addressees that the run stopped before
completion of the run
• Run Started – Notifies addressees that the run began
• Run Completed – Notifies addressees that the run is finished

4. In the “Enter email addresses for notifications” field, enter the e-mail address(es)
(including you) to which notifications are sent. Use the format shown on the
screen. Enter a comma between addresses.

5. Define the outgoing server. If you need information about the server, contact your
network system administrator.
a. In the Outgoing Server (SMTP) field, enter the name of the outgoing server.
For example: smtp.mycompany.com
b. Select Yes next to “Server requires an encrypted connection?” if the outgoing
server requires an encrypted connection. If no encrypted connection is
required, select No.
c. If the outgoing server requires authentication to receive the e-mail from the
instrument, select Yes next to “Server requires authentication?” Enter the
authentication user name and password in the dialog box.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 39

 Chapter 4 Run the Plate
4 Start or stop the run

Start or stop the run

IMPORTANT! If the computer that performs the run is on a network, avoid excess use
of the network during a run.

Note: You can set analysis parameters before or after you run a plate. To set
parameters before you run a plate, see Chapter 5, “Select Analysis Settings and
Thresholds”.

Start

To start a run:
• In the Experiment Menu, select Setup, select any screen,
then click Start Run at the top right corner.
or
• In the Experiment Menu, select Run, select any screen, then carefully click Start
Run at the top left corner.
Note: If you double-click the Start Run button, it may not become a Stop Run
button, but the run proceeds normally.

Stop
When you start a run, the green Start Run button becomes a red Stop
Run button. Click the Stop Run button to stop the run immediately.

Monitor the run (7500 System Only)

IMPORTANT! The following procedure is for the 7500 Real-Time PCR Instrument only
and not available for the QuantStudio™ 5 Real-Time PCR Instrument.

During a run, you can access the amplification plot, temperature plot, and run method.
In the Experiment Menu, select Run, then click:
• Amplification Plot – To view amplification plots of reactions
• Temperature Plot – To view temperature plots of reactions
• Run Method – To view and edit the run method during the run

40 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 4 Run the Plate
Save the results 4
Save the results
After a run is complete, HID Real-Time PCR Analysis Software automatically
performs analysis and saves the initial results file. If you modify the plate (for example,
if you remove a well from analysis and reanalyze the results), the software does not
automatically save the changes. After reanalysis, the HID Real-Time PCR Analysis
Software prompts you to save the results.
After the run, see Chapter 6, “Enhance Data Analysis,” to view and manage the
results.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 41

 Chapter 4 Run the Plate
4 Save the results

42 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 CHAPTER 5

Select Analysis Settings and

5 Thresholds

This chapter covers:

n Open analysis settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
n View/Edit CT settings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
n Enter HID settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
n Enter Flag settings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
n Add a virtual standard curve to the experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
IMPORTANT! All default settings shown in this guide and in the software screens are
for illustration only. For your experiments, set the parameters and thresholds
according to your laboratory protocol.

Before analyzing data from a completed run, you can edit values for the analysis
parameters:
• CT threshold, baseline start cycle, and end cycle
• HID flag thresholds
• QC flag thresholds
The Analysis Settings screen also contains the area where you set the parameters for
the Dilution Calculation tool to use in calculating a dilution scheme for downstream
amplification.
Note: See “Edit dilution settings for individual samples” on page 65 for more
information about settings in the Dilution Scheme area.

Open analysis settings

1. In the Experiment Menu, select Analysis, then select any one of the following
data displays:
• Amplification Plot
• Standard Curve
• Virtual Standard Curve
• Multicomponent Plot
• Raw Data Plot
• QC Summary

2. Click the Analysis Settings button in the top right corner of the
screen to display the Analysis Settings dialog box.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 43

 Chapter 5 Select Analysis Settings and Thresholds
5 View/Edit CT settings

View/Edit CT settings
Select the CT Settings tab to view the settings for CT. The recommended CT settings for
each Quantifiler™ kit are included in the experiment templates provided with the
software and in the user guide for the associated Quantifiler™ kit. The recommended
settings are those which were used in the validation experiments performed for each
kit by Thermo Fisher Scientific.
The default system settings are:
• Manual CT Threshold = 0.2
• Manual Baseline Start Cycle = 3
• Manual Baseline End Cycle = 15
To change these settings, click Edit Default Settings, then enter the new values.

To analyze the data with new settings, click Apply Analysis Settings at the bottom of
the Analysis Settings dialog box.

44 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 5 Select Analysis Settings and Thresholds
Enter HID settings 5
Enter HID settings

1. Select the HID Settings tab to view the Dilution Scheme, HID Flags, and HID
Flag settings.
See “Edit dilution settings for individual samples” on page 65 for more
information about settings in the Dilution Scheme area.

2. In the Use column in the HID Flags table, select the check box for each flag that
you want to include in the analysis.
You can use a flag to identify quality issues and help to interpret results for wells.
Flags can indicate samples that may require further attention. You can exclude
wells from data analysis. See “Omit wells from analysis” on page 55 for
instructions on excluding wells from analysis.

3. Enter threshold settings for the flag(s) that you select:

a. In the HID Flags table, select the flag of interest.
b. In the HID Flag Settings area, enter in the corresponding fields the value(s)
that you want to use.

Repeat steps 2 and 3 until you enter settings (or view the default settings), for all
the flags that you select.
Note: To save your HID flag settings for future use, save the experiment as a
template before you start the run (see “Start or stop the run” on page 40).

4. To analyze the data with new settings, click Apply Analysis Settings at the
bottom of the Analysis Settings dialog box.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 45

 Chapter 5 Select Analysis Settings and Thresholds
5 Enter HID settings

HIGHQT
The HIGHQT flag indicates that the quantity, or mean quantity of sample replicates, is
above a threshold that you set.

IPCCT
The IPCCT flag indicates one of the following:

Well
Cause Comment
contents

Unknown The IPC (Internal PCR Control) CT We strongly recommend that you base the threshold setting on
sample value is greater than the average of validation data produced by your laboratory. We have observed the
the IPC CT values for all the standards following:
plus the threshold that you set. • For information on interpreting the IPCCT flag for Quantifiler™
Kit experiments, refer to the user guide for the kit you are using.
Standard The IPC (Internal PCR Control) CT In Quantifiler™ Kit experiments, IPC target amplification should
or NTC value is above or below the maximum be within an expected range. Low or no IPC amplification can
or minimum, respectively, that you set. indicate the presence of PCR inhibitors, incorrect experiment
setup, or reagent or instrument failure.

LOWQT
The LOWQT flag indicates that the quantity, or mean quantity of sample replicates, is
below a threshold that you set.

NTCCT
This flag refers to the CT value of the NTC (non-template control). No amplification of
human and/or male target(s) should occur in NTC wells.

MTFR flag and M:F ratio display

The MTFR (Male to Female Ratio) is expressed as 1:X. A well is flagged if X is greater
than the threshold that you set. For example, if you set the MTFR flag threshold at 1:10,
then a sample containing 5 ng/µL of male DNA and more than 55 ng/µL of human
DNA generates an MTFR flag. The flag for this condition is a yellow triangle ( ) in
the Plate Layout or Well Table tab, and a red octagon ( ) in the Analysis Summary
(see Chapter 6, “Enhance Data Analysis”).
Samples that generate the MTFR flag are labeled “Thresholds Not Met” in the Analysis
Summary area of the QC Summary tab. The MTFR flag indicates samples that might
require Yfiler™ Kit amplification due to low quantities of male DNA relative to
female DNA. Autosomal amplification of these samples may result in partial to no
profile for the secondary (male) contributor.
In contrast, the M:F ratio display does not have an associated flag. The M:F ratio is also
expressed as 1:X and is displayed in the M:F ratio column of the well table only if X is
greater than or equal to the threshold that you set for the M:F ratio display.

46 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 5 Select Analysis Settings and Thresholds
Enter HID settings 5
The M:F ratio display threshold is expressed as 1:X where X must be less than or equal
to the X value for the MTFR flag. For example, if you set the M:F ratio display to 1:1,
then the MTFR flag must be set to 1:>1. Samples with ratios greater than the MTFR flag
display the MTFR flag and display the calculated M:F ratio. The M:F Ratio Display
function alerts you to male and female mixtures before STR analysis.

Table 3 Results of example M:F and MTFR settings

HID setting

Male Female Male:Female M:F Ratio M:F ratio MTFR

DNA DNA ratio MTFR flag display? flag?
display
(1:X) X =
(1:X) X =

1 ng/µL 1 ng/µL 1:1 1 1 Yes No

1 ng/µL 2 ng/µL 1:2 1 1 Yes Yes
1 ng/µL 1 ng/µL 1:1 1 2 Yes No

SLOPE
Indicates the PCR amplification efficiency for the experiment. The amplification
efficiency is calculated using the slope of the regression line in the standard curve. The
standard wells are flagged if the slope is not between the minimum and maximum
values that you set.
The standard curve is derived from a serial dilution set of standards containing a range
of known quantities. Results from amplifications of these standards are used to
generate a curve.
A slope of − 3.3 indicates 100% amplification efficiency. Refer to the Quantifiler™
Human DNA Quantification Kit and Quantifiler™ Y Human Male DNA Quantification Kit
User’s Manual and the Quantifiler™ Duo DNA Quantification Kit User’s Manual for more
information on the standard curve and slope.

R2
This flag indicates the regression coefficient calculated from the regression line of the
standard curve. The R2 value indicates the closeness of fit between the standard curve
regression line and individual CT data points from the standard reactions. A value of
1.00 indicates a perfect fit between the regression line and the data points.

YINT
The Y-intercept value of the standard curve indicates the expected CT value for a
sample with a quantity of 1 (for example, 1 ng/µL). The YINT flag can assist in
evaluating standard performance and serial dilution preparation. Your laboratory can
perform validation studies to determine a range for the Y-intercept and you can set
the HID Flag values for each Quantifiler™ kit and the HID Flag values for each target
(human and male) in the Quantifiler™ Duo assay. A YINT flag may indicate
incorrectly prepared standard concentrations, degraded standard, or other
preparation errors.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 47

 Chapter 5 Select Analysis Settings and Thresholds
5 Enter Flag settings

Enter Flag settings

1. Select the Flag Settings tab to view and define instrument, sample, and data
collection flags. Flags not used in the analysis are gray. Table 4 explains the flags.

2. In the Use column, select each flag that you want to include in the analysis.

3. Select the condition (< > =) in the Condition column drop-down lists and enter the
corresponding values in the Value column to specify the conditions that generate
a flag.

4. To omit the wells that have a flag from the analysis, select the corresponding
Reject Well check boxes.

5. To analyze the data with new settings, click Apply Analysis Settings.

Table 4 QC flags

Flag Description

AMPNC Amplification in non-template control

BADROX Bad passive reference signal
BLFAIL Baseline algorithm failed
CTFAIL CT algorithm failed
DRNMIN Define acceptable delta Rn based on Ct range
EXPFAIL Exponential algorithm failed
OFFSCALE Fluorescence is offscale
HIGHSD High standard deviation in replicate group
PRFLOW Low passive reference signal
NOAMP No amplification
NOISE Noise higher than others in plate
SPIKE Noise spikes
NOSIGNAL No signal in well
OUTLIERRG Outlier in replicate group
PRFDROP Passive reference signal changes near CT
THOLDFAIL Thresholding algorithm failed

48 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 5 Select Analysis Settings and Thresholds
Add a virtual standard curve to the experiment 5
Add a virtual standard curve to the experiment
If you are using a virtual standard curve to analyze experiments, use the software to
create the virtual standard curve, then assign it to your experiments as needed or
export it for further use.

Guidelines for using virtual standard curves

• The software will not analyze an experiment using a virtual standard curve if:
– The plate layout of the experiment contains wells that are configured with
the “standard” task type.
– The Expiration Date specified for the virtual standard curve has expired.
– The Quantifiler Kit specified for the experiment and the curve do not match.
• When analyzing an experiment using a virtual standard curve, all "unknown"
samples generates the IPCCT (Internal PCR Control Ct) flag by default.
• Laboratories should perform internal validation studies to ensure that
implementation of a virtual standard curve is appropriate and generates reliable
downstream data. For optimal results, virtual standard curves should be
evaluated independently for each real-time PCR instrument. We recommend the
re-evaluation of virtual standard curves with each new lot of quantification kit.

Create a virtual standard curve

IMPORTANT! To create the virtual standard curve, you must know the slopes and y-
intercepts of the targets for the kit that you are using.

1. In the Experiment Menu, select Analysis, then select Virtual Standard Curve.

2. Click the Add Standard Curve to Experiment button in the top left corner of the
screen to display the Virtual Standard Curve Library dialog box.

3. In the Virtual Standard Curve Library

dialog box, click New to create a new
virtual standard curve.

4. Specify the settings for the virtual

standard curve:
a. Enter a name for the curve.
b. (Optional) Select Is Standard
Curve Default? to analyze all
new experiments of the same
selected kit type (see substep 4d)
using the virtual curve.
c. Select the date on which the
curve expires. When the curve
expires, the software can no
longer use it to analyze data.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 49

 Chapter 5 Select Analysis Settings and Thresholds
5 Add a virtual standard curve to the experiment

d. Select the kit to which the virtual standard curve applies.

e. Enter the Slope and Y-Intercept for each target of the selected kit.
f. Enter any comments for the virtual standard curve, then click OK to save it
to the library.

Apply a virtual standard curve to an experiment

Before you apply the The software cannot use a virtual standard curve to analyze an experiment that
curve already contains wells that are assigned the Standard task. Therefore, before applying
a virtual standard curve, you must either omit or reassign the task of any well on the
plate layout that is configured as a standard.

To… See…

Omit wells from the analysis “Omit wells from analysis” on page 55
Reassign the task assignment of a well “Assign samples, standards, and NTCs to
wells” on page 30

Apply a standard IMPORTANT! Before you apply a virtual standard curve, you must either omit or
curve reassign the task of any well configured as a standard on the plate layout.

To apply a virtual standard curve to the open experiment:

1. In the Experiment Menu, select Analysis, then select Virtual Standard Curve.

2. Click the Add Standard Curve to Experiment button in the top left corner of the
screen.

3. From the Virtual Standard Curve Library dialog box, click Add selected Virtual
Standard Curve.

Note: When analyzing an experiment using a virtual standard curve, all "unknown"
samples generate the IPCCT (Internal PCR Control Ct) flag by default.

Automatic analysis If you select Is Standard Curve Default? in the

using a default settings of a virtual standard curve, then the HID
virtual standard Software automatically analyzes all new
experiments using that default virtual standard
curve
curve unless:
• The plate layout of a new experiment contains wells that are configured as
standards.
• The Expiration Date setting for the default virtual standard curve has passed.
• You select the Is Standard Curve Default? option for another virtual standard
curve (or the option is deselected for the existing virtual standard curve).

50 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 CHAPTER 6

Enhance Data Analysis

6
This chapter covers:
n View the analysis results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
n Interpret QC flag information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
n Omit wells from analysis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
n Omit targets in an experiment well . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
n Examine the Degradation Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
n Change the appearance of, print, and save plots . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 51

 Chapter 6 Enhance Data Analysis
6 View the analysis results

View the analysis results

Flagged wells

To view the results of the data analysis:

1. In the Experiment Menu, select AnalysisQC Summary to open the QC

Summary screen.
Note: If screen resolution is not set to 1280 X 1024, the Analysis Summary may
not be properly displayed.

2. In the QC Summary area, select the Analysis Summary tab to display areas that
list the HID-specific flags that you selected to include in the data analysis and
indicate the number of wells that meet/do not meet the threshold that you set. The
table below shows the meaning of the symbols.

Location Symbol Meaning

Standard Curve bar Green square ( ) A value for Slope, R2, or Y-

Intercept meets the
threshold
Red octagon ( ) A value for Slope, R2, or Y-
Intercept does not meet the
threshold
All Thresholds Met column of: Hyperlinked numbers The number of wells that
• Standard bar meet the thresholds for a
flag value
• NTC bar
• Unknown bar
All Thresholds Not Met column of: Hyperlinked numbers The number of wells that
• Standard bar do not meet the thresholds
for a flag value
• NTC bar
• Unknown bar

52 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 6 Enhance Data Analysis
View the analysis results 6
Standard curve The Standard Curve bar contains the SLOPE, R2, and Y-Intercept flags. Click the
column heading for a red octagon ( ) to highlight in the plate layout the wells
represented in the standard curve. This graphical view simplifies the identification of
wells that require further analysis using your laboratory protocol.

Standard The Standard bar reports the IPCCT flags for all the wells on the plate that you
designated as sample type Standard. Click the number in the Thresholds Not Met
column to view the well(s) that do not meet the IPCCT threshold in the plate layout or
well table format. You can use the amplification, multi-component, or the raw data
plot(s) to troubleshoot the data for these wells. You can examine the wells that meet the
threshold by clicking the number in the All Threshold Met column.

NTC (non-template The NTC bar reports the IPCCT and NTCCT flags for all the wells on the plate that you
control) designated as sample type NTC (non-template control). Click the number in the
Thresholds Not Met column to view the well(s) that do not meet the IPCCT or NTCCT
threshold in the plate layout or well table format. You can use the amplification, multi-
component, or raw data plot(s) to troubleshoot the data for these wells. You can
examine the wells that meet the threshold by clicking the number in the All Threshold
Met column.

Unknown The Unknown bar reports the IPCCT, HIGHQT, LOWQT, and MTFR flags for all the
wells on the plate that you designated as sample type Unknown (note that the MTFR
flag is not available in Human, HP, or Human Male kit experiments). The HIGHQT,
LOWQT, and MTFR (male to female ratio) flags indicate that the quantity of DNA or
ratios of male to female DNA in unknown samples might require additional attention.
Numbers below the flag indicate the number of wells that do not meet the threshold.
Click the number in the Thresholds Not Met column to view the well(s) that do not
meet a threshold in plate layout or well table format. You can use the amplification,
multi-component, or raw data plot(s) to troubleshoot the data for these wells. You can
examine the wells that meet the threshold by clicking the number in the All Threshold
Met column.

Instrument-related In addition to the flags listed above, a message might be displayed to indicate that one
flags or more of the instrument-related flags is generated by a potential problem with the
instrument. The message prompts you to select the QC Flags Details tab to view the
flags.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 53

 Chapter 6 Enhance Data Analysis
6 Interpret QC flag information

Well(s) automatically omitted

In certain rare instances, such as assignment of targets to empty wells, HID Real-Time
PCR Analysis Software may automatically omit certain wells of a Quantifiler™ Kit run.

The software automatically omits wells that may prevent the completion of data
analysis, so that analysis can continue for the rest of the wells in the plate. These wells
are indicated by a red exclamation point above the Analysis Summary tables. You can
examine the automatically omitted wells by clicking the number next to the
exclamation point.

Interpret QC flag information

QC Flags Detail

1. In the QC Summary screen, select the QC Flags Detail tab to view all QC flags
(both general and HID).

2. Click a flag to select all affected wells in the plate layout, and to open a brief
description of the flag and wells in a box below the list.

Also in the QC Flags Details description box is a hyperlink to online Help for
troubleshooting the flag and the criteria used for analysis (see Chapter 5, “Select
Analysis Settings and Thresholds,” for more information about these flags).
For more information about how to view and edit the information about samples, see
“Change the appearance of a plot” on page 57.

54 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 6 Enhance Data Analysis
Omit wells from analysis 6
Omit wells from analysis
You can omit wells from analysis. To view data from individual wells on the
Amplification analysis plot, in the Experiment Menu, select one of the following
screens:
• Amplification – Amplification vs. cycle and amplification vs. well
• Standard curve – CT vs. quantity of standards, flagged samples, and unflagged
samples
• Multicomponent plot – Fluorescence vs. cycle of all reaction components
• Raw data plot – Amplitude vs. filter
• Multiple plots view – Amplification, Standard curve, Multicomponent, and Raw
data plots in one pane

1. In the Experiment Menu, select Analysis. Click any Analysis screen. If no data are
displayed, click Analyze.

2. Omit wells using the well table or plate layout:

To use the well table, select the View Well Table tab, then select the Omit check
boxes corresponding to the wells to exclude from the analysis.
Note: If the Omit Well column is not visible in the table, click Show in Table, then
select Omit Well to show the column.

or
To use the plate layout, select the View Plate Layout tab. Right-click the well(s) to
omit, then select OmitWell.

3. Click Analyze to reanalyze the experiment data with the omitted well(s) excluded
from the analysis.

4. Review the data that are analyzed without the omitted well(s).

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 55

 Chapter 6 Enhance Data Analysis
6 Omit targets in an experiment well

Omit targets in an experiment well

For Duo, HP, and Trio experiments, you can omit one of the standard targets in a well
from analysis (shown for a Duo experiment in the example below).

1. Right-click a well with a standard target that you want to omit.

Note: You can omit only one target from one well at a time.

2. Select Omit from the drop-down list, then select:

• Well – to omit all targets from the well. The (well omitted) icon appears
in the well.
• Individual Target (for example, Duo Human) – to omit a specific target from
the well, select the name of the target. The individual target omitted icon (for
example, for Duo Human omitted) appears in the well.

3. Click Analyze to reanalyze the experiment data with the omitted target(s)
excluded from the analysis.

Examine the Degradation Index

Degradation Index refers to the data observed when a sample may be degraded: a
decrease in measured amount for large DNA fragments compared to small DNA
fragments. While DNA degradation is not the only theoretically possible mechanism
for a decrease in amount, it is the predominant mechanism in the absence of inhibitors.
The Degradation Index is for use as a general indicator of whether large DNA
fragments may perform more poorly in STR reactions. Evaluate Degradation Index in
conjunction with the IPC CT.
The Degradation Index is automatically calculated by the HID Real-Time PCR
Software using the following formula:
Small autosomal target DNA conc. (ng/μL)

Large autosomal target DNA conc. (ng/μL)

The Degradation Index value is displayed in the Well Table view in any of the analysis
screens (you may have to scroll to the right to display it.)
For more information on Degradation Index or evaluating the quality indices
determined by the HID Real-Time PCR Software to determine if highly degraded
samples can be better analyzed with the Ion Personal Genome Machine™ (PGM™)
System, refer to the Quantifiler™ HP and Trio DNA Quantification Kits User Guide.

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 Chapter 6 Enhance Data Analysis
Change the appearance of, print, and save plots 6
Change the appearance of, print, and save plots
Change the appearance of a plot
1. In the Experiment Menu, select Analysis, then click the name of a plot of interest.

2. In the plot screen, locate the icon bar above the plot.

3. Click (Hide) to hide the plot legend.

4. To change the appearance of a plot, click (Edit Plot Properties) to open the Plot
Properties dialog box. Three tabs are displayed.

5. Select the appropriate tab to enter the values you want to use to plot the data.

6. Click OK to apply the changes.

Specify wells to report

You can specify which wells to include in the amplifications plots and results table of
reports:

1. In the Experiment Menu, select any Analysis screen.

2. Select the well(s) to include, using either the View Plate Layout tab (see step 4 on
page 31) or the View Well Table tab (see step 4 on page 33).

Print or save a plot

Click (Print) to print the plot.
Click (Save) to save the plot as a *.jpg file.
Printed plots and *.jpg files include the slope, Y-intercept, and R2.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 57

 Chapter 6 Enhance Data Analysis
6 Change the appearance of, print, and save plots

58 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 CHAPTER 7

Export and Report Results

7
This chapter covers:
n Export data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
n Print a report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Overview
After the HID Real-Time PCR Analysis Software completes analysis and after you
review the data, you can generate a customized report in *.pdf files, then save or print
the report.
You can also export and save data in these formats:
• Excel™ (*.xls)
• Powerpoint™ (*.ppt)
• Text (*.txt)

Export data

1. In the Experiment Menu, select Analysis. Click any Analysis screen, then click
either View Plate Layout or View Well Table.

2. Highlight the wells to export.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 59

 Chapter 7 Export and Report Results
7 Export data

3. In the toolbar, click (Export) to open the Export Data screen, then select the
Export Properties tab.

4. Select the type of data to export:

• Amplification Data – Data that was collected during the cycling or
amplification stage.
• Multicomponent Data – Fluorescence data for each dye, for each cycle.
• Raw Data – Raw fluorescence data for each filter, for each cycle.
• Results – Results of the analysis.
• Sample Setup – Setup information such as well, sample name, and sample
color.
• STR Dilution Setup – Sample dilution worksheet to prepare samples for
amplification. For more information, see Chapter 8, “Generate Dilution and
Reaction Worksheets for STR Setup.”
• STR Reaction Setup – STR reaction setup worksheet to prepare samples for
amplification. For more information, see Chapter 8, “Generate Dilution and
Reaction Worksheets for STR Setup.”

5. Select Separate Files or One File in the drop-down list.

6. Enter the export file properties. For:

• Export File Name – Enter the name of the report.
• File Type – Select the type of file to which you want to send the data. Refer to
the online Help for information on creating *.ppt slides.
• Export File Location – Enter the filepath to the location where you want to
store the report.

7. To customize the data:

a. Select the Customize Export tab.

b. Select the information that you want to export.
Note: Sample setup should be exported as a .txt file only.

c. To sort data in the export by column, click the column header (for example,
click Well to sort the data by well).

8. Click Start Export to export the data to the file(s) that you selected.

60 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 7 Export and Report Results
Print a report 7
Print a report

1. Click Plate SetupAssign Targets and Samples, then click either View Plate
Layout or View Well Table.

2. Highlight the wells to include in the report.

3. In the toolbar, click (Print Report) to display the Print Report screen.

4. Select the check box corresponding to each data topic that you want to include in
the report.
Note: Exported standard curves do not include unknown data points.

5. Click Print Preview or Print Report at the bottom of the screen.

IMPORTANT! To save the report to a file, you must click Print Preview before you
print the report.

6. Select Save to save the report, or select Print to print the report.
Note: If you do not enter a name in the Experiment Name field of the Experiment
Properties screen, the experiment name on the report is “Untitled.”

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 61

 Chapter 7 Export and Report Results
7 Print a report

62 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 CHAPTER 8

Generate Dilution and Reaction

8 Worksheets for STR Setup

This chapter covers:

n Add kits to an experiment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
n Select unknown samples for amplification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
n Edit dilution settings for individual samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
n View the dilution scheme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
n Export dilution and reaction worksheets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
n Save new STR Kit information from an experiment into STR Kit Library. . . . . . 66

Overview
After a run is complete, you can use the HID Real-Time PCR Analysis Software to
generate dilution and reaction worksheets for STR set up.
The software generate dilution and reaction setup worksheets to perform calculations
for the kit(s) you select from the STR Kit Library, and the kit information and default
dilution settings you specify.
See Appendix A, “Configure STR Library and Default Dilution Settings” to:
• Enter, edit, or delete kit information in the STR Kit Library
• Set default dilution settings for the calculations

Add kits to an experiment

Before exporting worksheets, add kits to an experiment:

1. Open the experiment of interest.

2. In the Experiment Menu, select STR Kit Setup.

3. In the STR Kit Setup area, click Add Kit to Experiment to open the Kit Dilutions
Library.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 63

 Chapter 8 Generate Dilution and Reaction Worksheets for STR Setup
8 Select unknown samples for amplification

4. Select the kit(s) to use in the experiment. To edit kit information, see “Configure
the STR Kit Library” on page 73.

5. Repeat steps 2 through 4 until you select all the kits to use in the experiment.

6. To delete a kit from the experiment (not from the Kit Library), select the kit to
delete, then click Delete Kit from Experiment.

Select unknown samples for amplification

After adding kits to an experiment, select the unknown samples for amplification and
associate samples with kits:

1. In the Experiment Menu, select any analysis screen, then select the View Well
Table tab.
Note: If the Well Table does not display a column for the selected STR kit, click
Show in Table, then select the kit name from the list of available columns.

2. Select the check box corresponding to the unknown sample to

use and the STR kit with which to use the sample. If a sample
is not for amplification (for example a standard), it is not
available for selection.
To select all of the samples for a kit, select the check box beside
the kit name at the top of the column.
Note: The software automatically assigns the same kit for replicates.

3. Select the Dilution Setup tab to view the dilution scheme and the STR kit(s) that
you selected for each sample.

4. Repeat steps 2 and 3 for each unknown sample and kit(s).

Note: You cannot select an STR kit for standard or NTC sample types. Dilution
calculations apply only to the unknown sample (Human or Male) target in the well(s),
not to standards or NTCs.

64 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 8 Generate Dilution and Reaction Worksheets for STR Setup
Edit dilution settings for individual samples 8
Edit dilution settings for individual samples

If needed, edit the default dilution settings for samples:

1. Select the View Well Table tab.

2. Select the sample of interest.

3. In the toolbar at the top of the well table, click Edit Dilutions to open the Edit
Target Dilution Details screen.

Note: If you quantify replicates, this screen displays the sample concentration or
the mean sample concentration.

4. View or edit:
• Min. Pipetting Vol. – The minimum quantity to pipette.
• Max. Sample Vol. – The maximum volume of available sample.
• Dilution Factor – For example, enter 10 for 10-fold dilutions.
• Target Conc. – The amount of target DNA that you want to use divided by
the total sample volume per STR reaction.
• # Replicates – The number of identical reactions.

Note: The software displays target sample concentration based on maximum sample
volume, number of replicates, sample volume per STR reaction, and pipetting overage
that you set if the desired target concentration cannot be reached.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 65

 Chapter 8 Generate Dilution and Reaction Worksheets for STR Setup
8 View the dilution scheme

View the dilution scheme

View the dilution scheme to ensure settings are appropriate for the experiment:

1. In the Experiment Menu, select Analysis.

2. Click any plot to open a plot screen.

3. Select the Dilution Setup tab to open the Dilution Setup screen.

4. Review the dilution setup settings for downstream reactions.

Export dilution and reaction worksheets

Export the STR Dilution Setup worksheet and the STR Reaction Setup worksheet as
described in “Export data” on page 59.

Save new STR Kit information from an experiment into STR Kit
Library
You can save a kit from an experiment into the library (for example, if you import an
experiment from a system with a different library setup).
Note: If the STR kit name you are saving from the experiment is already listed in the
library, rename or delete the kit from the library before saving the kit information from
the experiment.

1. Open the experiment.

2. In the STR Kit Setup screen, select the kit to save.

3. Click Save Kit to Library.

66 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 CHAPTER 9

HID Real-Time PCR Analysis

9 Software Validation

This chapter covers:

n Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
n Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
n Experiments and results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
n Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

Introduction
The HID Real-Time PCR Analysis Software v1.3 is designed specifically for the
Quantifiler™ DNA Quantification Kits and the Applied Biosystems™ 7500 Real-Time
PCR System or the QuantStudio™ 5 Real-Time PCR System with 0.2-mL 96-well
sample block. The software enables streamlined quantification run setup, data
analysis, and STR reaction setup by providing Quantifiler™-specific templates and
quality flags as well as STR sample normalization (dilution) and reaction setup tools.
HID Real-Time PCR Analysis Software v1.3 contains the same functionality as the
version 1.2 software in addition to new features that support the use of virtual
standard curves and the use of the QuantStudio™ 5 system. See “Features in v1.3” on
page 10 for more information.
This chapter describes the results of experiments that Thermo Fisher Scientific
performed to validate the HID Real-Time PCR Analysis Software v1.3. Data was
collected using versions 1.2 and 1.3 of the HID Real-Time PCR Analysis Software, the
7500 Real-Time PCR System and the QuantStudio™ 5 Real-Time PCR System, and the
Quantifiler™ HP, Trio, Duo, and Human DNA Quantification Kits.
The data collected from both the 7500 and QuantStudio™ 5 instruments were
analyzed to verify:
• The HID Real-Time PCR Analysis Software v1.3 performs as designed to analyze
data generated on the 7500 Real-Time PCR System and the QuantStudio™ 5 Real-
Time PCR System.
• The new features do not adversely affect either the quantification assays or the
software functionality carried over from the HID Real-Time PCR Analysis
Software v 1.2.
• Data generated using the 7500 Real-Time PCR System and the QuantStudio™ 5
Real-Time PCR System when analyzed using the HID Software v1.3 demonstrate
reproducible performance for the respective instrument models.

For validation experiments and results for the Quantifiler™ Trio, Duo, HP, and
Human kits, see the Quantifiler™ HP and Trio DNA Quantification Kits User Guide
(Pub. no. 4485354), the Quantifiler™ Duo DNA Quantification Kit User Guide
(Pub. no. 4391294), and the Quantifiler™ Human and Y Human Male DNA
Quantification Kits User Guide (Pub. no. 4344790).

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 67

 Chapter 9 HID Real-Time PCR Analysis Software Validation
9 Materials and methods

Materials and methods

• Instrument, computer, and software configuration
– Applied Biosystems™ 7500 Real-Time PCR System, firmware v2.10 (3)
– QuantStudio™ 5 Real-Time PCR System, 0.2-mL 96-well sample block
(3)
Instrument 1 Instrument 2 Instrument 3 Instrument 4 Instrument 5 Instrument 6

QuantStudio 5 QuantStudio 5 QuantStudio 5

Model 7500 System 7500 System 7500 System
System System System
Computer Laptop Laptop Desktop Laptop Laptop Desktop
Microsoft™ OS Windows™ 7 64-bit Windows™ 7 32-bit
HID Real-Time PCR
v1.3 v1.2 and v1.3 v1.2
Analysis Software

• Chemistries and consumables

Item Cat. No.

Quantifiler™ Trio DNA Quantification Kit, 14 kits (from single lot) 4482910
Quantifiler™ HP DNA Quantification Kit, 5 kits (from single lot) 4482911
Quantifiler™ Duo DNA Quantification Kit, 5 kits (from single lot) 4387746
Quantifiler™ Human DNA Quantification Kit, 5 kits (from single lot) 4343895
MicroAmp™ Optical 96-Well Reaction Plate, 10 plates N8010560
Modified TE Buffer (10/0.1) 300675
MicroAmp™ Optical Adhesive Film, 100 covers 4311971
7500 Real-Time PCR Systems Spectral Calibration Kit I 4349180
ABY™ Dye Spectral Calibration Plate, 96-well 4461591
JUN™ Dye Spectral Calibration Plate, 96-well 4461593
TaqMan™ RNase P Instrument Verification Plate, 96-well 4350584
AmpFlSTR™ Control DNA 007, Male N/A
AmpFlSTR™ Control DNA 9947A, Female N/A

The following test cases were performed for each chemistry kit:

Plates per instrument for the experiment

Quantifiler™
Kit Precision/ Accuracy/
Sensitivity Mixture Inhibition
linearity reproducibility

Trio Kit 3 plates 1 plate 1 plate 1 plate 1 plate

Duo Kit 3 plates 1 plate 1 plate 1 plate 1 plate
HP Kit 1 plate 1 plate — — —
Human Kit 1 plate 1 plate — — —

68 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Chapter 9 HID Real-Time PCR Analysis Software Validation
Materials and methods 9
• Samples

Experiment Sample Replicates per plate

Precision and Quantifiler™ Trio, Duo, HP, and Human 6 standard curve
linearity Kit DNA Standards 12 dilution series
Accuracy and
One male DNA, 1 ng/µL 96 replicates
reproducibility
• Quantifiler™ Trio Kit – One male and • 5 dilution series
one female DNA diluted to 100, 10, 1, 0.1,
Sensitivity 0.01, 0.001, and 0.0001 ng/µL
• Quantifiler™ Duo Kit – One male DNA • 5 dilution series
diluted to 50, 5, 0.5, 0.05, and 0.005 ng/µL
• Quantifiler™ Trio Kit – One set of • 3 mixture series
male/female DNA mixture at 1:0, 1:1,
Mixture 1:10, 1:100, 1:1000, 1:4000, 0:1 ratios
analysis • Quantifiler™ Duo Kit – One set of • 3 mixture series
male/female DNA mixture at 1:0, 1:1,
1:10, 1:100, 1:500, 1:1000, 0:1 ratios
• Quantifiler™ Trio Kit – One male 007 • 96 replicates
DNA (0.1 ng/µL) with hematin (550 μM)
Inhibition
• Quantifiler™ Duo Kit – One male 007 • 96 replicates
DNA (0.1 ng/µL) with hematin (80 μM)

• Data collection and analysis

– Run Method and analysis settings were configured as outlined in the
respective Quantifiler™ kit user manuals. The design and execution of all
workflows (including instrument calibration, run setup, data analysis, run
methods, HID quality flags, dilution calculations import/export and
reporting) were identical for both versions of the software.
Note: The 7500 Systems were calibrated as explained in the Applied
Biosystems™ 7500/7500 Fast Real-Time PCR Systems System Maintenance Guide
(Pub. no. 4387777).
Note: The QuantStudio™ 5 Systems were calibrated as explained in the
QuantStudio™ 3 and 5 Real-Time PCR Systems Installation, Use, and Maintenance
Guide (Pub. no. MAN0010407). The instruments were calibrated for the ABY-
HID and JUN-HID dyes using the ABY™ Dye Spectral Calibration Plate (Cat.
no. 4461591) and the JUN™ Dye Spectral Calibration Plate (Cat. no. 4461593).
– Calibration and experiment (.eds) data files that were previously generated
using HID Real-Time PCR Analysis Software v1.2 were imported and
reanalyzed using HID Real-Time PCR Analysis Software v1.3. The results
from v1.2 and v1.3 were then compared for differences in data output.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 69

 Chapter 9 HID Real-Time PCR Analysis Software Validation
9 Experiments and results

Experiments and results

Instrument performance

Test Case Description Passing Criteria Results

Using each instrument, set up and run: For each respective system (7500
• Three plates of the standard curve dilution or QuantStudio™ 5):
series using the Quantifiler™ Trio and Duo • When running standard curves
Kits, standard curve dilution series. using the Quantifiler™ Trio, HP,
• One plate using the Quantifiler™ HP and DUO, and Human Assays, the CT
Human Kits, standard curve dilution series. values shall have a coefficient of
Precision and
variation (CV) ≤ 20% within an Pass
linearity Produce a standard curve from each pair of instrument.
dilution series, so that six curves are
• When running standard curves
generated per plate on each instrument.
using the Quantifiler™ Trio, HP,
Statistically evaluate the CT and R2 values for
DUO, and Human Assays, the
variation within an instrument.
system shall have a standard curve
R2 value ≥ 0.98.
Using each instrument, set up and run one When running the Quantifiler™ Trio,
plate of 007 DNA with 1 ng/µL input using each HP, DUO, and Human Assays, the
Accuracy and
Quantifiler™ Kit, 96 replicates per plate. within-instrument CT and quantity Pass
reproducibility
Statistically evaluate the CT values and DNA values shall have a CV ≤ 20% within an
quantity for variation within an instrument. instrument.
Using each instrument, set up and run: When running Quantifiler™ Trio Assays
• One plate containing seven dilutions of 007 using 0.01 ng/µL to 10 ng/µL DNA input
and 9947a DNA (from 0.0001 ng/µL to 100 and when running Quantifiler™ Duo
ng/µL) prepared using the Quantifiler™ Trio Assays using 0.05 ng/µL to 5 ng/µL
Kit. DNA input, the in-plate CT and quantity
values shall have a CV ≤ 20% within an
• One plate containing five dilutions of
007 DNA (from 0.005 ng/µL to 50 ng/µL) instrument.
Sensitivity Pass
prepared using the Quantifiler™ Duo Kit.
Run five dilution series per sample per plate,
providing a total of five replicates for each
sample dilution. Statistically evaluate the CT
values and DNA quantity data for variation
within an instrument.
Using each instrument, set up and run: When running Quantifiler™ Trio and
• One set of male and female DNA mixture DUO Assays using mixtures where the
samples consisting of seven mixture ratios male DNA input is 0.02 ng/µL and
(1:0, 1:1, 1:10, 1:100, 1:1000, 1:4000, and female DNA input is between 0.02 and
0:1) using the Quantifiler™ Trio Kit. 20 ng/µL, the CT and quantity values
shall have a CV ≤ 20% within an
• One set of male and female DNA mixture
Mixture instrument when results are within
samples with seven mixture ratios (1:0, 1:1, Pass
analysis range on the standard curve.
1:10, 1:100, 1:500, 1:1000, and 0:1) using the
Quantifiler™ Duo Kit.
Run three replicates for each mixture sample.
Statistically evaluate the CT values and DNA
quantity data for variation within an
instrument.

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 Chapter 9 HID Real-Time PCR Analysis Software Validation
Experiments and results 9
Test Case Description Passing Criteria Results

Using each instrument, set up and run: When running Quantifiler™ Trio and
• One plate of 0.1 ng/µL 007 DNA with 550 µM DUO Assays, the generated CT and
hematin using the Quantifiler™ Trio Kit. quantity values shall have a CV ≤ 20% Pass
within an instrument.
• One plate of 0.1 ng/µL 007 DNA with 80 µM Exception
Inhibition
hematin using the Quantifiler™ Duo Kit. conditions
1,2
Run 96 replicates per plate. Statistically
evaluate the CT values and quantity data for
variation within an instrument.
1 When tested using the Quantifiler™ DUO Kit and the QuantStudio™ 5 Instrument, samples with extreme Hematin concentrations (>40 µM) can
produce a biphasic curve that may result in overestimation of DNA concentrations or quantity value CV significantly >20%.
2 At extreme Hematin concentrations (approximately 550 µM), more variation (quantity value CV significantly >20%) may be observed in the large
autosomal and Y targets on both 7500 and QuantStudio™ 5 systems.

Software performance

Test Case Description Passing Criteria Results

Using one 7500 System and one QuantStudio™ The software shall successfully collect
5 System, set up a virtual standard curve the data and automatically apply the
using estimated slope and y-intercept values. virtual standard curve. The generated Pass
Perform a Quantifiler™ Trio run without DNA quantities shall be 100%
standard curve samples. concordant with the manual calculation.

Custom Using three 7500 Systems and three For each respective system (7500 or
standard QuantStudio™ 5 Systems, evaluate the virtual QuantStudio™ 5), analyzing the same
curve standard curve function using the sensitivity of a experiment using the actual and virtual
collected run. Create a virtual standard curve standard curves, the software shall
using the parameters (slope and y-intercept) of calculate quantification values that Pass
the standard curve generated from the run. match to the third decimal point.
Analyze the data using both the actual standard
curve and the virtual standard curve, then
compare the DNA quantity values.
Using one 7500 System computer and one When comparing the v1.2 and v1.3
QuantStudio™ 5 System computer, confirm that software, all values for the run/analysis
Pass
the settings match for all run/analysis methods methods and flag thresholds shall be
Workflow and and flag thresholds. same.
user interface Using both 7500 and QuantStudio™ 5 Systems, The software functions necessary for
run Quantifiler™ Trio and Quantifiler™ HP the use of the Quantifiler™ Assays
Pass
Assays by following the standard workflow and shall perform without error.
using the necessary software functions.
Use the v1.3 software to reanalyze an When analyzing the EDS file using the
experiment (EDS) file generated using a v1.2 and v1.3 software, the calculated
Backward
7500 System and the v1.2 software, then quantification values shall match to the Pass
compatibility
compare the quantification values calculated by third decimal point.
both versions of the software.

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 Chapter 9 HID Real-Time PCR Analysis Software Validation
9 Conclusions

Conclusions
Based on the validation of the HID Real-Time PCR Analysis Software v1.3 and the
precision and linearity, accuracy and reproducibility, sensitivity, mixture analysis, and
inhibition validation experiments performed using the Quantifiler™ Trio, Duo, HP, and
Human Kits [see the Quantifiler™ HP and Trio DNA Quantification Kits User Guide
(Pub. no. 4485354), Quantifiler™ Duo DNA Quantification Kit User Guide
(Pub. no. 4391294), and Quantifiler™ Human and Y Human Male DNA Quantification
Kits User Guide (Pub. no. 4344790)]:
• All updates to v1.3 were successfully and correctly implemented without
negative effects on functionality carried over to v1.3 from v1.2.
• The HID Real-Time PCR Analysis Software v1.3 successfully controlled both the
7500 Real-Time PCR Systems and QuantStudio™ 5 Real-Time PCR Systems, and
reliably and reproducibly set up and collected quantification data using the
Quantifiler™ kits.
• The software provided accurate results when used to process Quantifiler™ kits for
the analysis of genomic DNA samples.
• The user interface and HID workflows in v1.3 software performed as expected for
both instruments when using the Windows™ 7 operating systems.
• The coefficient of variation (CV) for the average values of DNA quantity data and
standard curve parameters (CT and R2 values) where tested varied less than 20%
within each instrument (7500 and QuantStudio™ 5 system) using both HID Real-
Time PCR Software versions 1.2 and 1.3.
Laboratories should determine the appropriate level of testing required before
implementation, based on the nature of the changes made to the software, how the
software pertains to their laboratory workflow, their internal software validation
guidelines, and those of the appropriate governing agencies.

72 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 APPENDIX A

Configure STR Library and Default

A Dilution Settings

Configure the STR Kit Library

Most AmpFlSTR Kits are listed in the STR Kit Library by default. To add or modify
amplification kit information:

1. In the toolbar, select ToolsAmpFlSTR Kit Library to open the Kit Dilutions
Library screen.

2. To:
• Add a kit – click New. The Create New STR Kit dialog box opens.
• Configure a kit – select the kit, then click Edit. The Create New STR Kit
dialog box opens.
• Remove a kit – select the kit, then select Delete.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 73

 Appendix A Configure STR Library and Default Dilution Settings
A Configure the STR Kit Library

3. Enter settings:
• STR Kit Name – The name of the kit that you are adding to the list.
Note: Kit names must be unique. To use the same kit with different
sample types or different input amounts of DNA, add the kit with a
different name, such as Identifiler_1.5 ng.
• Target Conc. – The amount of DNA that you want to use divided by the total
sample volume per reaction. Examples:

Total DNA (ng) Volume/reaction (µL) Target Conc. (ng/µL)

0.5 10 0.05
1.0 10 0.1
1.0 20 0.05
2.0 20 0.1

• STR Reaction
– PCR Master Mix – Enter appropriate volumes (µL)
– Sample – Enter appropriate volumes (µL)
The volume of the Master Mix volume and the volume of the sample
must equal the total volume of the STR reaction:
Sample (µL) + PCR Master Mix (µL) = Reaction Volume (µL)

• Additional # of Reactions and/or Amplification Controls – Enter the

number of additional STR reactions per amplifications to allow for pipetting
overage.
IMPORTANT! Because not all kits allow for pipetting overage, you might
need to enter more Additional Reactions to compensate for volume losses
that occur during pipetting. Refer to your kit user manual (see page 78) for
information about pipetting overage.
• PCR Master Mix – List each component of the STR Reaction Master Mix.
Refer to your kit user's manual for more information.

4. Click OK.

5. Repeat steps 2 through 4 for all needed kits.

6. Verify that the kits to be used in the downstream STR reactions are listed, with
correct information.
Note: You can also save a kit from an experiment into the library (for example, if
you import an experiment from a system with a different library setup). See “Save
new STR Kit information from an experiment into STR Kit Library” on page 66.

74 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Appendix A Configure STR Library and Default Dilution Settings
Configure the STR Kit Library A
Set default dilution settings

In Analysis Settings, you can specify default dilutions settings to apply to all samples
(You can edit individual sample dilution settings after you associate an STR kit with an
experiment).

1. In the Experiment Menu, select any analysis screen, then click Analysis Settings.

2. Select the HID Settings tab.

3. In the Dilution Method area, select:

• One Step Dilution – To use a single dilution in all instances.
or
• System Select – To use a dilution scheme that depends on your preferences,
with a maximum of two dilutions.
The software displays target sample concentration based on maximum sample
volume, number of replicates, sample volume per STR reaction, and pipetting
overage that you set if the desired target concentration cannot be reached.

4. In the Dilution Scheme area, enter dilution scheme parameters according to your
preferences or laboratory protocol. Enter the:
• Pipetting overage – The percent to add to compensate for error in pipetting.
If the sample concentration is less than the target concentration and the
sample volume is limited, set the pipetting overage to zero to maximize the
amount of DNA in the STR reaction.
• Minimum Pipetting Volume – The minimum volume that you want to
pipette.
• Maximum Sample Volume – The maximum quantity of sample that you
want to use.
• Dilution Factor – The maximum first dilution that you want to perform with
the available DNA. For example, for 10-fold first dilutions, enter 10.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 75

 Appendix A Configure STR Library and Default Dilution Settings
A Configure the STR Kit Library

76 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Documentation and Support

How to use your documentation

Portable document format (PDF) versions of this guide and the documents listed in
this section are available at http://www.thermofisher.com.
Note: To open the user documentation available from the Life Technologies web
site, use the Adobe™Acrobat™ Reader™ software available from www.adobe.com.

HID Real-Time PCR Analysis Software users

Refer to the following documents for more information about using HID Real-Time
PCR Analysis Software.

Product Title Purpose Pub. no.

Quantifiler™ Kits Quantifiler™ HP and Trio DNA Provides further information on DNA 4485354
Quantification Kit User Guide quantification of samples containing human
and male DNA using multiple-copy target
loci for improved detection sensitivity.
Quantifiler™ Duo DNA Quantification Kit Provides further information on DNA 4391294
User Guide quantification of samples containing mixed
human and male DNA
Quantifiler™ Human DNA Quantification Provides further information on DNA 4344790
Kit and Quantifiler™ Y Human Male quantification of samples containing
DNA Quantification Kit User Guide human/male DNA
7500/7500 Fast 7500/7500 Fast Real-Time PCR Provides information on instrument 4412844
Real-Time PCR Systems Maintenance Guide maintenance
Systems
7500/7500 Fast Real-Time PCR System Provides further information on system 4378658
Getting Started Guide for Absolute operation and data analysis
Quantification Experiments
7500/7500 Fast Real-Time PCR System Provides further information on system 4387779
Getting Started Guide for Standard operation and data analysis
Curve Experiments
QuantStudio™ 5 QuantStudio™ 3 and 5 Real-Time PCR Provides information on preparing, using, MAN0010407
Real-Time PCR Systems Installation, Use, and maintaining, and troubleshooting the
Systems Maintenance Guide system
QuantStudio™ Design and Analysis Provides instructions for performing MAN0010408
Desktop Software User Guide standard curve experiments on the
QuantStudio™ Design and Analysis desktop
Software

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 77

Documentation and Support
Obtaining related documentation

Documents for custom experiments

Refer to the following documents for information on performing custom experiments
instead of using HID Real-Time PCR Analysis Software:

7500/7500 Fast Real-Time PCR System Getting Started Guide for… Part Number

Genotyping Experiments 4387784

Presence/Absence Experiments 4387785
Relative Standard Curve and Comparative CT Experiments 4387783
Standard Curve Experiments 4387779
Applied Biosystems™ 7300/7500/7500 Fast Real-Time PCR 4378658
System Absolute Quantification Getting Started Guide

Obtaining related documentation

For more information on analysis methodology, refer to the 7500/7500 Fast Real-Time
PCR System Getting Started Guide for Standard Curve Experiments or the QuantStudio™
Design and Analysis Desktop Software User Guide.
For more information on PCR amplification kits, go to www.thermofisher.com.

Obtaining information from the Help system

The HID Real-Time PCR Analysis Software has a Help system that describes how to
use each feature of the user interface. Access the Help system by doing one of the
following:
• Click in the toolbar of the HID Real-Time PCR Analysis Software window.
• Select HelpContents and Index.
• Press F1.
You can use the Help system to find topics of interest by:
• Reviewing the table of contents
• Searching for a specific topic
• Searching an alphabetized index

78 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Documentation and Support
Customer and technical support

Customer and technical support

For HID support:
• In North America—Send an email to HIDTechSupport@lifetech.com, or call
888-821-4443 option 1.
• Outside North America—Contact your local support office.
• For latest services and support information for all locations, go to
thermofisher.com/support.

Limited product warranty

Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth
in the Life Technologies' General Terms and Conditions of Sale found on Life
Technologies' website at www.thermofisher.com/us/en/home/global/terms-and-
conditions.html. If you have any questions, please contact Life Technologies at
www.thermofisher.com/support.

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 79

Documentation and Support
Limited product warranty

80 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Index

Numerics color, change 29

computer requirements 11
7500 Real-Time PCR System
calibrating 14 concentration
firmware requirement 11 DNA 74
memory upgrade required for instruments STR kit dilutions 65
purchased before 2008 13 configure analysis settings 43
Create New Standard Curve screen 49
create, virtual standard curve 49
A
CT analysis settings 44
Amplification Plot screen (analysis) 55
CTFAIL flag 48
Amplification screen (analysis) 55
custom experiment
Amplification screen (run) 40
documents for 78
AMPNC flag 48 selecting 26
analysis customize the software 19
flagged wells 52
flags 45
NTC 53 D
omit wells 55 default, virtual standard curve 50
QC 52 define
standard curve 55 samples 28
standards 53 targets 29
unknown 53 virtual standard curve 50
analysis settings Degradation Index 56
CT 44 delete wells 55
flags 45 dilutions
HID 45 library, sample 74
Analysis Settings screen 43 samples, individual 65
analysis summary 52 STR 65
apply, virtual standard curve 50 DNA amount 74
documentation 77
B DRNMIN flag 48
BADROX flag 48 dyes, reporter 29
BLFAIL flag 48
E
C enable notifications 39
Calibration exclude wells from analysis 55
procedure 14, 16 Experiment Properties screen 27
required materials 14, 16 experiment template.See template, experiment
spectra examples 15, 17 Experiment type setting 49
upgrading from software v1.2.3 14 experiment workflow 24

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 81

Index

experiments, custom 10 HIGHQT flag 46

EXPFAIL flag 48 HIGHSD flag 48
Expiration Date setting, virtual standard curve 49 Home screen 25
export Human, Quantifiler experiment 26
*.pdf 59 hybrid, Quantifiler experiment 26
*.ppt 59
*.txt 59
*.xls 59
I
instrument.See 7500 Real-Time PCR System or
QuantStudio™ 5 Real-Time PCR System
F instrument-related flags 53
flag settings 48 IPCCT flag 46
flagged wells 52 Is Standard Curve Default? setting 49, 50
flags
AMPNC 48
analysis 52 K
BADROX 48 kits for HID assays 9
BLFAIL 48
CTFAIL 48 M
DRNMIN 48
male, Quantifiler experiment 26
EXPFAIL 48
HIGHQT 46 materials, not included with Quantifiler™ Kits 14, 16
HIGHSD 48 method, run 38
instrument-related 53 MF ratio 46
IPCCT 46 monitor run 40
MTFR, MF ratio 46 MTFR flag 46
NOAMP 48 MTFR flag vs. MF ratio 46
NOISE 48 Multicomponent Plot screen 55
NOSIGNAL 48 Multiple Plots screen 55
NTCCT 46
OFFSCALE 48
OUTLIERRG 48 N
PRFDROP 48 NOAMP flag 48
PRFLOW 48 NOISE flag 48
R2 47 NOSIGNAL flag 48
SLOPE 47 Notification Settings screen 39
SPIKE 48 notifications, SMTP 39
THOLDFAIL 48 NTC, analysis 53
YINT 47 NTCCT flag 46
flags, QC, detail 54

O
G
OFFSCALE flag 48
graphs. modify, print, save 57 omit wells from analysis 55
online Help. See Help system
H outgoing server 39
Help system, accessing 78 OUTLIERRG flag 48
HID flags 45
HID settings 45

82 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

 Index

P S
parameters, run 38 sample
plate layout assign to wells 30
save 34 define 28
save as template 20, 34 save
Plate Setup screen 29 plots 57
plots. modify, print and save 57 sample name 28
PRFDROP flag 48 screen resolution, required 11
PRFLOW flag 48 screens
print Amplification (analysis) 55
plot 57 Amplification (run) 40
report 61 Amplification Plot (analysis) 55
specific wells 57 Analysis Settings 43
Create New Standard Curve 49
Experiment Properties 27
Q Home 25
QC analysis Multicomponent Plot 55
Degradation Index 56 Multiple Plots 55
flags detail 54 Notification Settings 39
instrument-related flags 53 Plate Setup 29
NTC 53 QC Summary 52
standard curve 53 Raw Data 55
standards 53 Run Method (run) 40
QC Summary screen 52 Run Method (setup) 38
quantity, DNA 74 Standard Curve 55
QuantStudio™ 5 Real-Time PCR System STR Kit Setup 63
calibration 16 Temperature Plot (run) 40
SDSs 79
server, outgoing 39
R
settings
R2 flag 47 dilution 65
ratio, MF 46 flag 45, 48
Raw Data screen 55 HID 45
report notification 39
export 59 virtual standard curve 49
print 61 Slope setting 49
reporter dyes 29 SLOPE, detail 47
resolution, screen, required 11 SMTP 39
run SPIKE flag 48
method 38 standard curve analysis 53
monitor 40
Standard Curve screen 55
start 40
standard curve, virtual 49
stop 40
standards
Run Method screen (run) 40
analysis 53
Run Method screen (setup) 38
assign to wells 30
start run 40
stop run 40

HID Real-Time PCR Analysis Software v1.3 Getting Started Guide 83

Index

STR 65
kit library 73
reaction volumes 74
STR Kit Setup screen 63

T
targets
assign to wells 30
concentration 74
define 29
Temperature Plot screen (run) 40
template, experiment
create 20, 34
link to home screen 20, 35
modify default 19
save plate layout 20
thermal profile 38
Thermo Fisher Scientific support 79
THOLDFAIL flag 48
thresholds, setting 43
training, information on 79

U
unknown, analysis 53

V
virtual standard curve
add to an experiment 49
apply 50
create 49
guidelines for use 49
set as default 50

W
wells, assign standards, targets, samples 30
workflow 24

Y
YINT flag 47
Y-intercept setting 49

84 HID Real-Time PCR Analysis Software v1.3 Getting Started Guide

thermofisher.com/support | thermofisher.com/askaquestion
thermofisher.com

27 August 2018