Materials and methods
9
Materials and methods
Experiments and results
Software • HID Real-Time PCR Analysis Software v1.1 (for data compatibility and concordance) • HID Real-Time PCR Analysis Software v1.2
Operating systems • Microsoft® Windows® XP 32 bit • Microsoft® Windows® 7 32 bit • Microsoft® Windows® 7 64 bit
Samples Quantifiler® Standard DNA, DNA Standard 007, 9947A, and 2 genomic DNA samples (male and female)
Instruments Two Applied Biosystems® 7500 Real-Time PCR Systems with thermal cycler firmware G2.10.
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.
• Calibration and .eds data files previously generated in HID Real-Time PCR Analysis Software v1.1 were imported and reanalyzed in HID Real-Time PCR Analysis Software v1.2. The results from v1.1 and v1.2 were then compared to determine whether there were any differences in data output.
Experiment Description Configuration, samples, and/or
files used Result
Install/uninstall/ upgrade testing
Test each installation pathway (v1.2 fresh installation and v1.2 upgrade installation from v1.1) to confirm that the installation process correctly installs, uninstalls (including Calibration, Experiment and Log file backup), and upgrades the software as required.
Test the upgrade installation pathway to verify that calibration files previously generated with HID Real-Time PCR Analysis Software v1.1 can be correctly imported and applied to data analyzed in HID Real-Time PCR Analysis Software v1.2.
• v1.2 fresh installation (tested using Quantifiler® Trio, HP, Duo, and Human kits)
• v1.1 upgrade to v1.2 (tested using Quantifiler® Trio, Duo, and Human kits)
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HID Real-Time PCR Analysis Software v1.2 Getting Started Guide
Chapter 9 HID Real-Time PCR Analysis Software Validation
Experiments and results
9
User interface testing
Set up and run Quantifiler® Trio, HP, Duo, and Human kits to verify the continued functionality of the user interface, including: menu items, windows, and file handling associated with instrument calibration, run setup, run methods, and run monitoring, instrument control, data analysis, plot/table views, STR reaction setup, STR dilution setup, file importing/exporting, and report generation.
To verify user interface functionality the following data were run and analyzed:
• Quantifiler® Duo kit (8 runs with a total of 672 samples and 3 .eds files containing a total of 18 samples) • Quantifiler® Human kit (2 runs with
a total of 192 samples)
• Quantifiler® Trio kit (11 .eds files containing a total of 131 samples) • Quantifiler® HP kit (6 .eds files
containing a total of 62 samples)
Pass
Functionality/ feature testing
Set up and run Quantifiler® Trio, HP, Duo, and Human kits, and use .eds files generated with HID Real-Time PCR Analysis Software v1.1, to verify that new software features and those carried over from v1.1 function properly and generate accurate results. The software functionality testing included: • Instrument calibration
• Run setup [includes the following: Use all new Quantifiler® templates, create run methods, assign samples to a plate and define targets, and import plate records (.txt)]
• Run monitoring
• Data analysis [includes the following: Enter, edit, and view HID flag settings, confirm flag results are correctly displayed, and omit targets and confirm reanalyzed data is accurate]
• STR reaction and dilution setup • File import/export
• Report generation
Verify accuracy and reliability of the following results: CT, quantity, HID-specific quality flags, M:F mixture ratios, Degradation Index, and STR reaction and dilution setup values.
To verify that existing and new features are functioning properly, the following data was run and analyzed:
• Quantifiler® Duo kit (11 runs with a total of 672 samples and 3 .eds files containing a total of 18 samples) • Quantifiler® Human kit (2 runs with
a total of 192 samples)
• Quantifiler® Trio kit (11 .eds files with a total of 131 samples) • Quantifiler® HP kit (6 .eds files
containing a total of 62 samples) The following data were used to verify the accuracy of the M:F ratio, dilution and STR setup and Degradation Index calculations:
• M:F calculations: Quantifiler® Duo and Trio kits (25 samples)
• Dilution calculations: Quantifiler® Duo, Trio, and HP kits (211 samples)
• Degradation Index calculations: Quantifiler® Trio and HP kits (193 samples)
Pass
Experiment Description Configuration, samples, and/or
Chapter 9 HID Real-Time PCR Analysis Software Validation
Conclusions
9
Conclusions
Based on the validation of the HID Real-Time PCR Analysis Software v1.2 and the developmental validation experiments performed for Quantifiler® Trio and HP Kits [Refer to Quantifiler® HP and Trio DNA Quantification Kits User Guide
(Pub. no. 4485354)]:
• All updates to v1.2 were successfully and correctly implemented without deleterious effects on functionality carried over to v1.2 from v1.1 (including accurately and reproducibly calculating the Degradation Index value for samples processed with Quantifiler® Trio and HP kits).
• The HID Real-Time PCR Analysis Software v1.2 properly calibrated and
controlled the 7500 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 v1.2 software behaved as expected when using both Windows® XP and Windows® 7 operating systems.
• 100% data concordance was observed for runs generated using HID Real-Time PCR Analysis Software v1.1 and re-analyzed using v1.2 (including CT values, standard curves, quantities, quality flags, as well as M:F mixture ratio and STR reaction and dilution setup calculations).
Laboratories should determine the appropriate level of testing that may be required Data concordance
and compatibility testing with HID Real-Time PCR Analysis Software v1.1
Verify templates and plates from HID Real- Time PCR Analysis Software v1.1 can be imported and experimental properties are retained and can be used for run setup, instrument control, and data collection in HID Real-Time PCR Analysis Software v1.2. Confirm data concordance between v1.2 and v1.1 using legacy Quantifiler® kit data generated in HID Real-Time PCR Analysis Software v1.1.
Data were generated using v1.1 and imported and re-analyzed in v1.2 to verify experimental properties are retained and data is concordant: Quantifiler® Duo, Human, Male, and Male & Human templates and 14 .eds files were examined.
Data used to verify the concordance of the M:F ratio and dilution/STR setup calculations:
• M:F ratio calculation: 4 v1.1 .eds files with a total of 142 samples • Dilution calculation and STR setup
worksheet: 10 v1.1 .eds files with a total of 60 samples
Pass
Experiment Description Configuration, samples, and/or
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HID Real-Time PCR Analysis Software v1.2 Getting Started Guide
A
APPENDIX A
Configure STR Library and Default
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 ToolsAmpFlSTR 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.
Appendix A Configure STR Library and Default Dilution Settings
Configure the STR Kit Library
A
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:
• 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:
• 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 74) 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 64.
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
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HID Real-Time PCR Analysis Software v1.2 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 yourpreferences 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.
Appendix A Configure STR Library and Default Dilution Settings
Configure the STR Kit Library
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HID Real-Time PCR Analysis Software v1.2 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 www.lifetechnologies.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.
Title Purpose Pub. no.
Quantifiler® HP and Trio DNA
Quantification Kit User Guide
Provides further information on DNA quantification of samples containing human and male DNA using multiple-copy target loci for improved detection sensitivity.
4485354
Quantifiler® Duo DNA
Quantification Kit User Guide
Provides further information on DNA quantification of samples containing mixed human and male DNA
4391294
Quantifiler® Human DNA
Quantification Kit and Quantifiler® Y Human Male
DNA Quantification Kit User Guide
Provides further information on DNA quantification of samples containing human/ male DNA
4344790
7500/7500 Fast Real-Time PCR Systems Maintenance Guide
Provides information on instrument maintenance
4412844
7500/7500 Fast Real-Time PCR System Getting Started Guide for Absolute
Quantification Experiments
Provides further information on system operation and data analysis
4378658
7500/7500 Fast Real-Time PCR System Getting Started Guide for Standard Curve Experiments
Provides further information on system operation and data analysis
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:
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.
For more information on PCR amplification kits, go to www.lifetechnologies.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 HelpContents 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
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 System Absolute Quantification Getting Started Guide
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HID Real-Time PCR Analysis Software v1.2 Getting Started Guide
Documentation and Support
Obtaining support
Obtaining support
For HID support:
• In North America - send an email to [email protected] or call 888-821-4443 option 1.
• Outside North America - contact your local support office. For the latest services and support information for all locations, go to:
www.lifetechnologies.com
At the Life Technologies web site, you can:
• Access worldwide telephone and fax numbers to contact Life Technologies Technical Support and Sales facilities.
• Search through frequently asked questions (FAQs). • Submit a question directly to Technical Support.
• Order Life Technologies user documents, SDSs, certificates of analysis, and other related documents.
• Download PDF documents.
• Obtain information about customer training. • Download software updates and patches.
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.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support.
Documentation and Support
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HID Real-Time PCR Analysis Software v1.2 Getting Started Guide
Index
Numerics
7500 Real-Time PCR System calibrating 13
firmware requirement 11
memory upgrade required for instruments pur- chased before 2008 12
A
Amplification Plot screen (analysis) 53 Amplification screen (analysis) 53 Amplification screen (run) 38 AMPNC flag 47 analysis flagged wells 50 flags 43 NTC 51 omit wells 53 QC 50 standard curve 53 standards 51 unknown 51 analysis settings CT 42 flags 43 HID 43
Analysis Settings screen 42 analysis summary 50
Applied Biosystems support 75
B
BADROX flag 47 BLFAIL flag 47C
Calibration procedure 13 required materials 13 spectra examples 14upgrading from software v1.2.3 13
color, change 27
computer requirements 10 concentration
DNA 70
STR kit dilutions 63 configure analysis settings 41 CT analysis settings 42 CTFAIL flag 47 custom experiment
documents for 74 selecting 24
customize the software 17
D
define samples 26 targets 27 Degradation Index 54 delete wells 53 dilutions library, sample 70 samples, individual 63 STR 63 DNA amount 70 documentation 73 dyes, reporter 27E
enable notifications 37exclude wells from analysis 53 Experiment Properties screen 25
experiment template.See template, experiment experiment workflow 22 experiments, custom 9 EXPFAIL flag 47 export *.pdf 57 *.ppt 57 *.txt 57 *.xls 57
Index
F
flag settings 46 flagged wells 50 flags AMPNC 47 analysis 50 BADROX 47 BLFAIL 47 CTFAIL 47 EXPFAIL 47 HIGHQT 44 HIGHSD 47 instrument-related 51 IPCCT 44 MTFR, MF ratio 44 NOAMP 47 NOISE 47 NOSIGNAL 47 NTCCT 44 OFFSCALE 47 OUTLIERRG 47 R2 45 SLOPE 45 SPIKE 47 THOLDFAIL 47 YINT 45 flags, QC, detail 52G
graphs. modify, print, save 55
H
Help system, accessing 74 HID flags 43
HID settings 43 HIGHQT flag 44 HIGHSD flag 47 Home screen 23
Human, Quantifiler experiment 24
K
kits for HID assays 9
M
male, Quantifiler experiment 24
materials, not included with Quantifiler® Kits 13
method, run 35 MF ratio 44 monitor run 38 MTFR flag 44
MTFR flag vs. MF ratio 44 Multicomponent Plot screen 53 Multiple Plots screen 53
N
NOAMP flag 47 NOISE flag 47 NOSIGNAL flag 47
Notification Settings screen 37 notifications, SMTP 37
NTC, analysis 51 NTCCT flag 44
O
OFFSCALE flag 47
omit wells from analysis 53 online Help. See Help system outgoing server 37 OUTLIERRG flag 47
P
parameters, run 35 plate layout save 32 save as template 18,
32 Plate Setup screen 2779
HID Real-Time PCR Analysis Software v1.2 Getting Started Guide
Index
Q
QC analysis Degradation Index 54 flags detail 52 instrument-related flags 51 NTC 51 standard curve 51 standards 51 QC Summary screen 50 quantity, DNA 70R
R2 flag 45 ratio, MF 44 Raw Data screen 53 reportexport 57 print 59 reporter dyes 27
resolution, screen, required 11 run
method 35 monitor 38 start 38 stop 38
Run Method screen (run) 38 Run Method screen (setup) 35
S
sample assign to wells 28 define 26 save plots 55 sample name 26screen resolution, required 11 screens
Amplification (analysis) 53 Amplification (run) 38
Amplification Plot (analysis) 53 Analysis Settings 42 Experiment Properties 25 Home 23 Multicomponent Plot 53 Multiple Plots 53 Notification Settings 37 Plate Setup 27 QC Summary 50 Raw Data 53
Run Method (run) 38 Run Method (setup) 35 Standard Curve 53 STR Kit Setup 61
Temperature Plot (run) 38 SDSs 75 server, outgoing 37 settings dilution 63 flag 43
,
46 HID 43 notification 37 SLOPE, detail 45 SMTP 37 SPIKE flag 47standard curve analysis 51 Standard Curve screen 53 standards analysis 51 assign to wells 28 start run 38 stop run 38 STR 63 kit library 69 reaction volumes 70 STR Kit Setup screen 61
T
targets
assign to wells 28 concentration 70 define 27
Temperature Plot screen (run) 38 template, experiment
create 18
,
32link to home screen 18
,
33 modify default 17save plate layout 18 thermal profile 35 THOLDFAIL flag 47 thresholds, setting 41 training, information on 75
U
unknown, analysis 51Index
W
wells, assign standards, targets, samples 28 workflow 22