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February 2021
Investigator® 24plex QS Handbook
For multiplex amplification of the CODIS core loci, the European standard set of loci, plus SE33, DYS391, and Amelogenin
Sample to Insight__
Contents
Kit Contents................................................................................................................ 3 Shipping and Storage ................................................................................................. 4 Intended Use .............................................................................................................. 4 Safety Information....................................................................................................... 5 Quality Control........................................................................................................... 5 Introduction ................................................................................................................ 6 Equipment and Reagents to Be Supplied by User ............................................................ 9 Protocol: PCR Amplification........................................................................................ 11 Protocol: Electrophoresis Using the Applied Biosystems 3500/3500xL Genetic Analyzer ........ 14 Protocol: Analysis ..................................................................................................... 29
Analysis Software ........................................................................................... 29 Controls ........................................................................................................ 30 Quality Sensor ............................................................................................... 31 Troubleshooting Guide .............................................................................................. 38 References ............................................................................................................... 40 Appendix A: Interpretation of Results........................................................................... 41 Appendix B: Varying PCR Volumes Using Investigator 24plex QS Kit.............................. 42 Ordering Information ................................................................................................ 43 Document Revision History ......................................................................................... 45
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Kit Contents
Investigator 24plex QS Kit Catalog no. Number of 25 µl reactions Fast Reaction Mix 2.0* Nuclease-free water Primer Mix 24plex QS Control DNA 9948 (0.1 ng/µl) DNA size standard 24plex (BTO) Allelic ladder 24plex Quick-Start Protocol
(100) 382415 100 750 µl 1.9 ml 250 µl 200 µl 55 µl 25 µl 1
* Contains DNA Polymerase, dNTPs, MgCl2, and bovine serum albumin (BSA).
(400) 382417 400 4 x 750 µl 4 x 1.9 ml 4 x 250 µl 200 µl 220 µl 3 x 25 µl 1
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Shipping and Storage
The Investigator 24plex QS Kit is shipped on dry ice. It should be stored immediately upon receipt at 30 to 15°C in a constant-temperature freezer. Avoid repeated thawing and freezing. The Primer Mix and allelic ladder must be stored protected from the light. DNA samples and post-PCR reagents (allelic ladder and DNA size standard) should be stored separately from the PCR reagents. Under these conditions, the components are stable until the expiration date indicated on the kit.
Once opened, the Investigator 24plex QS Kit should be stored at 28°C for a maximum of 6 months.
Intended Use
The Investigator 24plex QS Kit is intended for molecular biology applications in forensic, human identity, and paternity testing. This product is not intended for the diagnosis, prevention or treatment of a disease.
All due care and attention should be exercised in the handling of the products. We recommend all users of QIAGEN® products to adhere to the NIH guidelines that have been developed for recombinant DNA experiments or to other applicable guidelines.
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Safety Information
When working with chemicals, always wear a suitable lab coat, disposable gloves, and protective goggles. For more information, please consult the appropriate safety data sheets (SDSs). These are available online in convenient and compact PDF format at www.qiagen.com/safety, where you can find, view, and print the SDS for each QIAGEN kit and kit component.
Quality Control
In accordance with QIAGEN's ISO-certified Quality Management System, each lot of the Investigator 24plex QS Kit is tested against predetermined specifications to ensure consistent product quality. Investigator 24plex QS Kits meet ISO 18385 requirements.
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Introduction
The Investigator 24plex QS Kit is used for multiplex PCR in forensic, human identity, and paternity testing. The PCR simultaneously amplifies the 22 polymorphic STR markers listed below along with the gender-specific marker Amelogenin. These 22 markers are recommended by the CODIS (Combined DNA Index System) Core Loci Working Group, the European Network of Forensic Science Institutes (ENFSI) and the European DNA Profiling Group (EDNAP).
The Investigator 24plex QS Kit Primer Mix contains two innovative internal PCR controls (Quality Sensor QS1 and QS2) to provide helpful information about the efficiency of the PCR and the presence of PCR inhibitors. The Quality Sensors are amplified simultaneously with the polymorphic STR markers.
The Investigator 24plex QS Kit is specifically designed for rapid and reliable generation of DNA profiles from blood, buccal swabs, and forensic stains. The kit utilizes QIAGEN's fastcycling PCR technology, allowing amplification in around 60 minutes. It provides highly robust results with inhibitor-resistant chemistry. The primers are fluorescence-labeled with the following dyes:
6-FAMTM: Amelogenin, TH01, D3S1358, vWA, D21S11 BTG: TPOX, DYS391, D1S1656, D12S391, SE33 BTY: D10S1248, D22S1045, D19S433, D8S1179, D2S1338 BTR2: D2S441, D18S51, FGA BTP: QS1, D16S539, CSF1PO, D13S317, D5S818, D7S820, QS2
The recommended amount of DNA under standard conditions is 0.5 ng. Internal validations demonstrated robust and balanced results with 0.22 ng DNA and reliable results with <0.1 ng DNA.
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The Investigator 24plex QS Kit was validated using the GeneAmp® PCR System 9700 (with Gold-plated 96-Well Silver Block) and the Applied Biosystems® 3500TM Genetic Analyzer.
Table 1 shows the STR loci with their chromosomal mapping and repeat motifs, which are concordant with the International Society for Forensic Genetics (ISFG) guidelines for the use of microsatellite markers (1).
For information about known microvariants not contained in the Investigator 24plex allelic ladder, see the National Institute of Standards and Technology (NIST) website (www.cstl.nist.gov/biotech/strbase/).
Table 1. Locus-specific information of the Investigator 24plex QS Kit
Locus Amelogenin X Amelogenin Y DYS391
D1S1656
D2S441 D2S1338 D3S1358 D5S818 D7S820 D8S1179 D10S1248
D12S391
D13S317
GenBank® accession number M55418 M55419 AC011302
NC_000001.9
AL079112 G08202 11449919 G08446 G08616 G08710 AL391869
G08921
G09017
Repeat motif of the reference allele
[TCTA]11 [TAGA]16 [TGA][TAGA][TAGG]1[TG]5 [TCTA]12 [TGCC]6[TTCC]11 TCTA [TCTG]2 [TCTA]15 [AGAT]11 [GATA]12 [TCTA]12 [GGAA]13 [AGAT]5 GAT [AGAT]7 [AGAC]6 AGAT [TATC]13
Chromosomal mapping Xp22.1-22.3 Yp11.2 Yq11.21
1q42
2p14 2q35 3p25.3 5q23.2 7q21.11 8q23.1-23.2 10q26.3
12p13.2
13q31.1
Table continues on the next page
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Table continued from the previous page
Locus D16S539 D18S51
GenBank accession number
G07925 L18333
D19S433
G08036
D21S11
D22S1045 CSF1PO FGA (FIBRA)
SE33 (ACTBP2) TH01 (TC11) TPOX vWA
AP000433
AL022314 X14720 M64982
NG000840 D00269 M68651 M25858
Repeat motif of the reference allele
[GATA]11
[AGAA]13
AAGG [AAAG] AAGG TAGG [AAGG]11
[TCTA]4 [TCTG]6 [TCTA]3 TA [TCTA]3 TCA [TCTA]2 TCCATA [TCTA]11
[ATT]14 ACT [ATT]2
[AGAT]12
[TTTC]3 TTTTTTCT [CTTT]13 CTCC [TTCC]2
[AAAG]9 AA [AAAG]16
[TCAT]9
[AATG]11
TCTA [TCTG]4 [TCTA]13
Chromosomal mapping 16q24.1 18q21.3 19q12
21q21.1
22q12.3 5q33.1 4q28.2
6q14.2 11p15.5 2p25.3 12p13.31
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Equipment and Reagents to Be Supplied by User
When working with chemicals, always wear a suitable lab coat, disposable gloves and protective goggles. For more information, consult the appropriate safety data sheets (SDSs), available from the product supplier.
All protocols
Hi-DiTM Formamide, 25 ml (Applied Biosystems cat. no. 4311320) Matrix Standards BT6 for multi-capillary instruments, e.g., 3500 Genetic Analyzers (see
the Ordering Information) Pipettes and pipette tips One of the following DNA analyzers:*
Applied Biosystems 3500 Genetic Analyzer Applied Biosystems 3500xL Genetic Analyzer One of the following PCR thermal cyclers:* QIAamplifier® 96 GeneAmp PCR System 9700 VeritiTM 96-Well Thermal Cycler ProFlexTM 96-well PCR System Bio-Rad® PTC-200 Biometra UNO-Thermoblock Eppendorf® Mastercycler® ep PCR tubes or plates Microcentrifuge for PCR tubes or plates
* This is not a complete list of suppliers and does not include many important vendors of biological supplies.
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Validity of analysis software for human identification products
Investigator Human Identification PCR Kits require calibration with an allelic ladder. Therefore, the software used must be compatible with human identification products for forensic applications. We recommend GeneMapper® ID-X Software. The Investigator Template Files facilitate data analysis and are valid with this software.
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Protocol: PCR Amplification
This protocol is for PCR amplification of STR loci from forensic samples using the Investigator 24plex QS Kit.
Important points before starting
Set up all reaction mixtures in an area separate from that used for DNA isolation and PCR product analysis (post-PCR analysis).
Use disposable tips containing hydrophobic filters to minimize cross-contamination risks. The recommended amount of DNA under standard conditions is 0.5 ng. Internal
validations demonstrated robust and balanced results with 0.22 ng DNA and reliable results with <0.1 ng DNA.
Things to do before starting
Before opening the tubes containing PCR components, vortex and then centrifuge briefly to collect the contents at the bottom of the tubes.
Procedure
1. Thaw PCR components and template nucleic acid. Mix thoroughly. Centrifuge briefly before use.
2. Prepare a master mix according to Table 2. The master mix contains all of the components needed for PCR except the template (sample) DNA and nuclease-free water. As some loss of reagents can occur during transfers, prepare the mix with additional reactions included. Also include positive and negative control reactions.
3. Vortex the master mix thoroughly, centrifuge briefly, and dispense appropriate volumes into PCR tubes or the wells of a PCR plate.
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4. Add template DNA and nuclease-free water to the master mix to give a final sample volume of 25 µl.
5. Prepare positive and negative controls. Positive control: Use 5 µl of the Control DNA (i.e., 500 pg). Negative control: Use nuclease-free water instead of template DNA in the reaction.
Table 2. Reaction setup
Component Fast Reaction Mix 2.0 Primer Mix Nuclease-free water (added in step 4) Template DNA (added in step 4) Total volume
Volume per reaction 7.5 µl 2.5 µl Variable Variable 25 µl
6. If template DNA was pipetted onto the rim or lid of the PCR tube, then centrifuge briefly to collect the contents at the bottom of the tubes.
7. Program the thermal cycler according to the manufacturer's instructions, using the conditions outlined in Table 3.
Note: If using the GeneAmp PCR System 9700 with an Aluminum Block, use "Std Mode", or with a Silver 96-Well Block or Gold-plated Silver 96 Well Block, use "Max Mode". Do not use "9600 Emulation Mode". 8. After the cycling protocol is completed, store samples at 30 to 15°C protected from the light, or proceed directly to electrophoresis.
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Table 3a. Standard cycling protocol
Temperature
98°C* 64°C 72°C 96°C 61°C 72°C 68°C 60°C 10°C
Time
30 s 55 s 5 s 10 s 55 s 5 s 5 min 5 min
* Hot-start to activate DNA polymerase.
Table 3b. Optional cycling protocol
Temperature
98°C* 64°C 72°C 96°C 61°C 72°C 68°C 60°C 10°C
Time
30 s 55 s 5 s 10 s 55 s 5 s 2 min 2 min
* Hot-start to activate DNA polymerase.
Number of cycles 3 cycles
27 cycles
Number of cycles 3 cycles
27 cycles
Table 3b details previously published cycling conditions which may continue to be used if incomplete adenylation is not visible within the electropherograms.
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Protocol: Electrophoresis Using the Applied Biosystems 3500/3500xL Genetic Analyzer
The Investigator 24plex QS Kit is validated for use on the 3500/3500XL Genetic Analyzer, which requires the following software: 3500 Data Collection Software v1 or v2 HID Updater 3500 Data Collection v2.0
Note: The user must be logged on to the PC as local administrator or with equivalent access rights to allow data to be written to the appropriate files.
For detailed instructions on instrument setup, spectral calibration or application of the Applied Biosystems 3500 Series Data Collection Software v1 or v2 and the GeneMapper ID-X Software v1.2, refer to the Applied Biosystems 3500/3500xL Genetic Analyzers User Guide.
The system with 8 capillaries is the Applied Biosystems 3500 Genetic Analyzer. The system with 24 capillaries is the Applied Biosystems 3500xL Genetic Analyzer.
The virtual filter set AnyDye is used for combined application of the 6 fluorescent labels 6FAM, BTG, BTY, BTR2, BTP, and BTO. This matrix standard is BT6.
The materials required for electrophoresis are given in Table 4.
Table 4. Materials required for electrophoresis
Material Capillary Polymer Buffer
Specifications
36 cm Array for Applied Biosystems 3500/3500xL Genetic Analyzer POP-4® for Applied Biosystems 3500/3500xL Genetic Analyzer Anode Buffer Container (ABC) 3500 Series Cathode Buffer Container (CBC) 3500 Series
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Spectral calibration/matrix generation
Before conducting DNA fragment size analysis, perform a spectral calibration with the 6 fluorescent labels 6-FAM, BTG, BTY, BTR2, BTP, and BTO for each analyzer (Table 5). The calibration procedure creates a matrix that is used to correct the overlapping of the fluorescence emission spectra of the dyes.
Important: Spectral calibration must be performed for each new capillary array. It comprises the following steps: Preparation of the instrument Preparation of the standard calibration plate Plate assembly and loading into the instrument Software setup of dye set BT6 Performing a spectral calibration run Checking the matrix
Preparation of the instrument
Before the spectral calibration process, ensure that the spatial calibration has been performed. This process is described in detail in the Applied Biosystems 3500/3500xL Genetic Analyzers User Guide.
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Table 5. The 6 fluorescent labels of BT6
Color Blue (B) Green (G) Yellow (Y) Red (R) Purple (P) Orange (O)
Matrix standard 6-FAM BTG BTY BTR2 BTP BTO
Preparation of the standard calibration plate for 8 capillaries (Applied Biosystems 3500 Genetic Analyzer)
1. Before opening the tubes, vortex and then centrifuge briefly to collect the contents at the bottom of the tubes.
2. Set up a mixture of formamide and Matrix Standard BT6 according Table 6.
Table 6. Setup of formamide and Matrix Standard BT6 mixture for 8 capillaries
Component Hi-Di Formamide Matrix Standard BT6 multi cap.
Volume 90 µl 10 µl
3. Vortex and then centrifuge the mixture briefly. 4. Load 10 µl of the mixture into each of the 8 wells in a 96-well plate at positions A1H1. 5. Denature for 3 min at 95°C. 6. Snap freeze by placing the plate on ice for 3 min.
A thermal cycler set to 4°C may be used to cool the plate instead.
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Preparation of the standard calibration plate for 24 capillaries (Applied Biosystems 3500xL Genetic Analyzer)
7. Before opening the tubes, vortex and then centrifuge briefly to collect the contents at the bottom of the tubes.
8. Set up a mixture of formamide and Matrix Standard BT6 according to Table 7.
Table 7. Setup of formamide and Matrix Standard BT6 mixture for 24 capillaries
Component Hi-Di Formamide Matrix Standard BT6 multi cap.
Volume 225 µl 25 µl
9. Vortex and then centrifuge the mixture briefly. 10.Load 10 µl of the mixture into each of the 24 wells in a 96-well plate at positions A1H1,
A2H2, and A3H3. 11.Denature for 3 min at 95°C. 12.Snap freeze by placing the plate on ice for 3 min.
A thermal cycler set to 4°C may be used to cool the plate instead.
Plate assembly and loading the plate in the instrument The necessary steps are described in detail in the Applied Biosystems 3500/3500xL Genetic Analyzers User Guide.
Software setup of dye set BT6
Prior to the spectral calibration, a dye set for the Matrix Standard BT6 must be set up.
1. To create a new dye set, select "Library". Under "Analyze" go to "Dye Sets" and click "Create".
2. Enter a "Dye Set Name", for example, BT6.
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3. Under "Chemistry" select "Matrix Standard" and as a "dye set template" select "AnyDye Template".
4. Under "Calibration Peak Order" arrange the colors as follows: 6 blue, 5 orange, 4 green, 3 yellow, 2 red, and 1 purple.
Note: This is the correct instrument setting of the peak order, although the peak order of the Matrix Standard BT6 is different. 5. Alter the "Parameters" settings as follows: Matrix Condition Number Upper Limit: 13.5 Locate Start Point After Scan: 1000 Locate Start Point Before Scan: 5000 Limit Scans To: 2750 Sensitivity: 0.4 Minimum Quality Score: 0.95
6. Click "Save" to confirm the changes.
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Figure 1. Setup of dye set BT6.
Performing a spectral calibration run
Once the multiwell plates containing the spectral calibration mixture are placed in the autosampler tray, the spectral calibration process can be started.
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7. To access the Spectral Calibration screen, select "Maintenance" on the Dashboard of the 3500 Series Data Collection Software.
8. To set up a calibration run, go to "Calibrate", followed by "Spectral" and select "Calibration Run".
9. The number of wells in the spectral calibration plate and the position in the instrument must be specified.
10.Under "Chemistry Standard" select "Matrix Standard", and as a "Dye Set" select the previously created BT6 (see "Software setup of dye set BT6", page 17).
11.(Optional) Enable "Allow Borrowing". 12.Click "Start Run".
Checking the matrix
Click a capillary in the table to display the results for each capillary below the run results table (Capillary, Quality value, and Condition Number).
The quality value (Q value) of each capillary must be greater than 0.95 and the condition number range (C value) must be between 1 and 13.5.
Check the matrix samples for a flat baseline. As shown in Figure 2, there should be 6 peaks with peak heights of about 10006000 RFU for each matrix sample. (Note: The optimal range is 30005000 RFU.)
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Figure 2. Electropherogram of spectral calibration of the matrix standard BT6 on an Applied Biosystems 3500 Genetic Analyzer.
When a spectral calibration is successfully completed, the "Overall" row displays green results (Figure 3). If the "Overall" row displays red results, refer to the "Spectral calibration troubleshooting" section of the Applied Biosystems 3500/3500xL Genetic Analyzers User Guide.
Figure 3. Example of successful spectral calibration of the matrix standard BT6 for all capillaries on an Applied Biosystems 3500 Genetic Analyzer.
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For each capillary, select and display the spectral and raw data. Check that the data meet the following criteria: The order of the peaks in the spectral profile from left to right should read orangered
yellowgreenbluepurple. No extraneous peaks should appear in the raw data profile. Peak morphology in the spectral profile should show no gross overlaps, dips or other
irregularities. Separate and distinct peaks should be visible.
If the data for all capillaries meet the criteria above, click "Accept". If any capillary data do not meet the criteria above, click "Reject" and refer to the "Spectral calibration troubleshooting" section of the Applied Biosystems 3500/3500xL Genetic Analyzers User Guide.
Sample preparation
1. Before opening the tubes, vortex and then centrifuge briefly to collect the contents at the bottom of the tubes.
2. Set up a mixture of formamide and DNA size standard according to Table 8. 3. Vortex and then centrifuge the mixture briefly. 4. Aliquot 12 µl of the mixture to a tube for each sample you plan to analyze. 5. Add 1 µl PCR product or allelic ladder (diluted, if necessary). 6. Denature for 3 min at 95°C. 7. Snap freeze by placing the plate on ice for 3 min. 8. A thermal cycler set to 4°C may be used to cool the plate instead. 9. Load the samples on the tray.
Table 8. Setup of formamide and DNA size standard mixture
Component Hi-Di Formamide DNA Size Standard 24plex (BTO)
Volume per sample 12.0 µl 0.5 µl
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Note: Since injections take place simultaneously on all capillaries, a minimum of 1 entire column (8-sample protocol) or 3 entire columns (24-sample protocol) must be pipetted onto the plate of multi-capillary analyzers. If fewer samples are analyzed, the empty positions must be filled with 12 µl Hi-Di Formamide. To ensure a reliable allelic assignment on multi-capillary analyzers, inject one allelic ladder for each set of 24 samples: 8-capillary instruments: One allelic ladder per 3 injections 24-capillary instruments: One allelic ladder per injection
Important: The actual room temperature may influence the performance of PCR products on multicapillary instruments, so shoulder peaks or split peaks can occur, especially at lower temperatures. Ensure that the ambient conditions are maintained as recommended by the instrument manufacturer. Also, ensure buffers are equilibrated to ambient conditions.
Setting up a run
If you are using the Investigator 24plex QS Kit for the first time on an Applied Biosystems 3500 Genetic Analyzer, you will first need to set up a number of protocols: Instrument Protocol Size Standard QC Protocol Assay
All protocols can be set up via the Dashboard of the 3500 Series Data Collection Software.
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Instrument Protocol
1. To set up the Instrument Protocol, select "Library", and then under "Analyze" go to "Instrument Protocols" and click "Create". Note: Modify the Run Module Default settings from "HID36_POP4" as shown in Table 9.
2. The parameters from Table 9 must be entered or selected. 3. Click "Save" to confirm the changes.
Table 9. Instrument Protocol parameters for Applied Biosystems 3500/3500xL Genetic Analyzer
Parameter Application Type Capillary Length Polymer Dye Set Run Module Protocol Name Oven Temperature (°C) Run Voltage (kV) PreRun Voltage (kV) Injection Voltage (kV) Run Time (s) PreRun Time (s) Injection Time (s) Data Delay (s) Advanced Options
3500 Setting HID 36 cm POP4 e.g., BT6 HID36_POP4 e.g., Investigator 24plex Default (60) 13.0 Default (15) 1.2 1550 Default (180) 30.0* Default (1) Default
3500xL Setting HID 36 cm POP4 e.g., BT6 HID36_POP4 e.g., Investigator 24plex Default (60) 13.0 Default (15) 1.6 1550 Default (180) 33.0* Default (1) Default
* Deviating from the standard settings, the injection time may range between 1 and 35 s depending on the type of sample. If samples with very high signal intensities are recorded, a shorter injection time may be selected. For samples with low DNA content, an injection time of up to 35 s may be necessary.
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Size Standard
4. To set up the Size Standard, select "Library", and then under "Analyze" go to "Size Standards" and click "Create".
5. The parameters in Table 10 must be entered or selected. The DNA Size Standard 24plex (BTO) should be used with the following lengths of fragments: 60, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 250, 260, 280, 300, 320, 340, 360, 380, 400, 425, 450, 475, 500, 525, and 550 bp.
Table 10. Size standard parameters Parameter Size Standard Dye Color
Setting e.g., SST-BTO_60-500bp Orange
6. Alternatively, import the DNA Size Standard 24plex (BTO) parameters using the recommended Investigator template file "SST-BTO_60-500bp" (Table 15).
7. Click "Save" to confirm the changes.
QC Protocol
8. To set up the QC Protocol, select "Library", and then under "Analyze" go to "QC Protocols" and click "Create".
9. The parameters in Table 11 must be entered or selected.
Table 11. QC Protocol parameters
Parameter Protocol Name Size Standard Sizecaller
Setting e.g., BTO_550 SST-BTO_60-500bp SizeCaller v1.1.0
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10. Go to "Analysis Settings", followed by "Peak Amplitude Threshold", and ensure that all colors are enabled.
Check the recommended analysis settings in Table 14. All other settings should remain as "Default". 11. Click "Save" to confirm the changes.
Assay
12. To set up an Assay, go to "Library", and then under "Manage" go to "Assays" and click "Create".
13. To analyze Investigator 24plex fragments, the parameters in Table 12 must be selected. 14. Click "Save" to confirm the changes.
Table 12. Assay parameters Parameter Assay Name Color Application Type Instrument Protocol QC Protocols
Setting e.g., Investigator 24plex Default HID e.g., Investigator 24plex e.g., BTO_550
Starting the run
15. In the Dashboard, click "Create New Plate". 16. Go to "Setup", followed by "Define Plate Properties", and select "Plate Details". Select
or enter the parameters in Table 13.
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Table 13. Plate properties Property Name Number of Wells Plate Type Capillary Length Polymer
Setting e.g., Investigator 24plex 96 HID 36 cm POP4
17. Click "Assign Plate Contents" to implement the changes.
18. Enter the designated sample name in each well containing a sample or allelic ladder. This will identify the well positions of each sample for the data collection and processing.
19. Under "Assay", choose the correct Assay for the analysis. If you followed the steps under "Setting up a run" (see page 23 onward), click "Add from Library" and select Investigator 24plex from "as Instrument Protocol". All named wells on the plate must have an assigned assay.
20. Repeat for "File name conventions" and "Results group".
21. Select the wells for which to specify an assay. Tick the boxes that are next to the names of "Assay", "File name conventions", and "Results group" to assign those to the selected wells.
22. If not already done, load the assembled plate to the instrument and close the instrument door to reinitialize the instrument. Then click "Link Plate for Run". In the next screen, enter the desired Run Name and click "Start Run".
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Analysis parameters/analysis method
Table 14 lists the recommended analysis parameters in the worksheet Peak Detector.
Table 14. Recommended settings for the Applied Biosystems 3500/3500xL Genetic Analyzer
Parameter
Settings
Peak Detection Algorithm
Advanced
Ranges
Analysis: Partial Range Start Point: 1000; Stop Point: 20,000 Sizing: All Sizes
Smoothing and Baselining
Smoothing: Light Baseline Window: 51 pts
Size Calling Method
Local Southern Method
Peak Detection
Peak Amplitude Thresholds B:* Y:* G:* R:* P:* O:* Min. Peak Half Width: 2 pts Polynomial Degree: 3 Peak Window Size: 11 pts Slope Thresholds: 0.0
* The peak amplitude threshold (cutoff value) corresponds to the minimum peak height that will be detected by the GeneMapper ID-X Software. The thresholds are usually 50200 RFU and should be determined individually by the laboratory. Recommendation: The minimal peak height should be three times higher than the background noise of the baseline.
Only the setting for Peak Window Size is different to the Applied Biosystems defaults for HID analysis.
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Protocol: Analysis
For general instructions on automatic sample analysis, refer to the appropriate User Guides for GeneMapper ID-X Software. Finding the exact lengths of the amplified products depends on the device type, the conditions of electrophoresis and the DNA size standard used. Due to the complexity of some loci, determining the size should be based on evenly distributed references. The DNA Size Standard 24plex (BTO) should be used with the following lengths of fragments: 60, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 250, 260, 280, 300, 320, 340, 360, 380, 400, 425, 450, 475, 500, 525, and 550 bp.
Figure 4. Electropherogram of the DNA Size Standard 24plex (BTO). Fragments lengths in bp.
Analysis Software
Allele allocation should be carried out using suitable analysis software e.g., GeneMapper ID-X Software in combination with the Investigator Template Files, which are available for download from www.qiagen.com.
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Table 15. Recommended Investigator Template Files for GeneMapper ID-X
File type Panels* BinSets* Stutter Size standard Analysis Method
File name 24plex_Panels_x 24plex_Bins_x 24plex_Stutter_x SST-BTO_60500bp Analysis_HID_3500_50rfu Analysis_HID_3500_200rfu
Plot Settings
Plots_6dyes
* Panels and Bin Sets must always be used; other template files are optional.
Controls
The alleles listed in Table 16 represent the Control DNA 9948 (included in the Investigator 24plex QS Kit) and DNA from other commercially available standard cell lines.
For further confirmation, Table 16 displays the alleles of reference DNA purchased from Coriell Cell Repositories (CCR), as well as 2 reference DNAs purchased from CCR to the standard of Szibor et al. (2).
Table 16. Allele assignment of the Investigator 24plex QS Kit
Locus Amelogenin DYS391 D1S1656 D2S441 D2S1338
CCR 9948 X/Y 10/10 14/17 11/12 23/23
CCR 9947A X/X 18.3/18.3 10/14 19/23
CCR 3657 X/Y 10/10 13/18.3 14/14 18/22
Table continues on the next page
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Table continued from the previous page
Locus D3S1358 D5S818 D7S820 D8S1179 D10S1248 D12S391 D13S317 D16S539 D18S51 D19S433 D21S11 D22S1045 CSF1PO FGA SE33 TH01 TPOX vWA
CCR 9948
15/17 11/13 11/11 12/13 12/15 18/24 11/11 11/11 15/18 13/14 29/30 16/18 10/11 24/26 23.2/26.2 6/9.3 8/9 17/17
CCR 9947A
14/15 11/11 10/11 13/13 13/15 18/20 11/11 11/12 15/19 14/15 30/30 11/14 10/12 23/24 19/29.2 8/9.3 8/8 17/18
CCR 3657
16/18 11/11 10/11 15/16 14/16 18/19 11/13 13/13 12/20 13/14 28/29 11/17 11/11 18/23 22.2/27.2 7/9.3 8/11 14/19
Quality Sensor
The Investigator 24plex QS Kit contains two internal PCR controls (Quality Sensor QS1 and QS2), which provide helpful information about the PCR amplification efficiency in general and about the presence of PCR inhibitors. The internal Quality Sensors are enclosed in the Primer Mix and are amplified simultaneously with the polymorphic STR markers. The Quality Sensors are labeled with BTP and appear at fragment sizes of 74 bp (QS1) and 435 bp (QS2).
To address the issue of sequence similarity and the possibility of non-specific binding, a synthetic internal control DNA template was designed using a random algorithm. The template sequence differs from all known DNA sequences, and in particular bears no similarity to human
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DNA. The chance of non-specific binding in the context of a multiplex PCR amplification reaction is therefore very low.
In general, the successful amplification of the small Quality Sensor (QS1) indicates that the PCR was set up and conducted correctly, regardless of whether DNA was present or absent in the sample. If no Quality Sensor is detected in the analysis of the amplification products, this means that either the pipetting during PCR setup or the PCR itself was performed incorrectly. This indicates that the user could repeat the experiment for improved results.
Sensitivity experiments have revealed that the internal controls have no effect on the performance of the PCR. The amplification of low DNA template amounts showed similar results for primer mixes with or without the Quality Sensors.
In addition, the analysis of the two internal control fragments, QS1 and QS2, and of the STR target amplification products allow for the differential identification of the presence of inhibitors or the presence of DNA degradation in an amplification reaction.
In the case of sample degradation, the amplification of smaller target fragments is more efficient than the amplification of larger target fragments. However, degradation of the target template does not hamper amplification of the internal control fragments from the internal control template (Figure 5). Thus, an equal ratio of QS1 and QS2, together with a ratio in favor of small STR target products, suggests the presence of sample degradation.
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Figure 5. Electropherogram of STR analysis in the presence of degraded DNA (fragments of 150 bp). Genomic DNA was sheared to fragments of 150 bp using ultrasound. The large STR fragments were amplified with a very low PCR yield, but QS1 and QS2 were amplified normally with equal peak heights. The markers are given at the top of the electropherogram. The Quality Sensors are labeled with BTP (panel 5) and appear at fragment sizes of 74 bp (QS1) and 435 bp (QS2).
If inhibitors such as hematin and humic acid are present in the sample, amplification is less efficient and larger DNA fragments are amplified less than smaller ones. If the analysis of the amplification products indicates an inefficient amplification of the larger STR target sequences and the larger Quality Sensor (QS2) fragment, but the smaller Quality Sensor (QS1) is amplified successfully, the sample is likely to have been contaminated with inhibitors. This means that a shift of the ratio in favor of the small Quality Sensor (QS1) suggests the presence of inhibitors (Figure 6).
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Figure 6. Electropherogram of STR analysis in the presence of hematin. 22 STR markers, Amelogenin, and the two Quality Sensors were amplified in the presence of 1000 µM hematin and analyzed using capillary electrophoresis. The amplification of high molecular weight fragments, including STR makers of more than 250 bp and QS2, was inhibited by the high hematin content. The markers are presented at the top of the electropherogram. The Quality Sensors are labeled with BTP (panel 5) and appear at fragment sizes of 74 bp (QS1) and 435 bp (QS2, not visible).
Analysis of the presence of the two Quality Sensors allows the user to differentially identify the presence of PCR inhibitors or the occurrence of degradation in the forensic sample. This gives the user helpful information for data interpretation and planning the next steps. Table 17 summarizes the possible profile appearances and their meanings.
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Table 17. Profile appearances and their meanings.
Allele Peaks
QS1
Present
Present
Absent
Present
Absent
Absent
Ski-slope profile
Present
Ski-slope profile
Present
QS2 Present Present Absent Dropdown Present
Interpretation Successful profile No DNA Failed PCR Inhibitors present Degraded DNA
Note: The peak heights of QS1 and QS2 may vary slightly between different experiments. A slight peak height scattering is usual, and is not dependent on inhibitor influence. During the validation the analyst should evaluate the usual variation spectrum in relation to their certain samples type, and should define a regular peak height range for both QS.
A drop down of the QS2 signal below 20% of the QS1 signal indicates inhibition of the PCR reaction.
Alleles
Table 18 shows the alleles of the allelic ladder. All analyses were performed using POP-4 polymer (Table 18 and Figure 7). Different analysis instruments, DNA size standards, or polymers may result in different fragment lengths. In addition, a visual alignment with the allelic ladder is recommended.
Scaling
Horizontal: 70470 bp Vertical: Depending on signal intensity
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Figure 7. Electropherogram of the allelic ladder 24plex analyzed on an Applied Biosystems 3500 Genetic Analyzer. The allelic ladder contains two alleles for each Quality Sensor (QS1 and QS2). This allows an automated calling of the QS peaks for sample analysis.
Table 18. Allelic ladder fragments included in the allelic ladder 24plex
Locus Amelogenin TH01 D3S1358 vWA
D21S11
Dye label 6-FAM 6-FAM 6-FAM 6-FAM
6-FAM
Repeat numbers of allelic ladder
X, Y 4, 5, 6, 7, 8, 9, 9.3, 10, 10.3, 11, 13, 13.3 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 24, 24.2, 25, 26, 26.2, 27, 28, 28.2, 29, 29.2, 30, 30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 35.2, 36, 36.2, 37, 38
Table continues on the next page
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Table continued from the previous page
Locus TPOX DYS391
Dye label BTG BTG
D1S1656
BTG
D12S391
BTG
SE33
BTG
D10S1248 D22S1045 D19S433
D8S1179 D2S1338 D2S441 D18S51
FGA
BTY BTY BTY
BTY BTY BTR2 BTR2
BTR2
QS1
BTP
D16S539
BTP
CSF1PO
BTP
D13S317
BTP
D5S818
BTP
D7S820
BTP
QS2
BTP
Repeat numbers of allelic ladder
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15
7, 8, 9, 10, 11, 12, 13
10, 11, 12, 13, 14, 14.3, 15, 15.3, 16, 16.3, 17, 17.3, 18, 18.3, 19.3, 20.3
14, 15, 16, 17, 17.3, 18, 18.3, 19, 20, 21, 22, 23, 24, 25, 26, 27
3, 4.2, 6.3, 8, 9, 10, 11, 12, 13, 13.2, 14, 14.2, 15, 15.2, 16, 17, 18, 18.2, 19, 19.2, 20, 20.2, 21, 21.2, 22, 22.2, 23.2, 24.2, 25, 25.2, 26.2, 27.2, 28.2, 29.2, 30.2, 31, 31.2, 32, 32.2, 33, 33.2, 34, 34.2, 35, 36, 36.2, 37, 38, 39, 42
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
6.2, 8, 9, 10, 11, 12, 12.2, 13, 13.2, 14, 14.2, 15, 15.2, 16, 16.2, 17, 17.2, 18.2
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19
12, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28
8, 9, 10, 11, 11.3, 12, 13, 14, 15, 16, 17
8, 9, 10, 10.2, 11, 12, 13, 13.2, 14, 14.2, 15, 16, 17, 17.2, 18, 18.2, 19, 20, 21, 21.2, 22, 23, 24, 25, 26, 27, 28
14, 16, 17, 18, 18.2, 19, 19.2, 20, 20.2, 21, 21.2, 22, 22.2, 23, 23.2, 24, 24.2, 25, 25.2, 26, 27, 28, 29, 30, 30.2, 31.2, 33, 34, 37.2, 42.2, 43.2, 44.2, 45.2, 46.2, 47.2, 48.2, 50.2, 51.2
Q, S
5, 8, 9, 10, 11, 12, 13, 14, 15
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18
5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16
Q, S
For information about known microvariants not contained in the Investigator 24plex allelic ladder, see the National Institute of Standards and Technology (NIST) website (www.cstl.nist.gov/biotech/strbase/).
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Troubleshooting Guide
This troubleshooting guide may be helpful in solving any problems that may arise. For more information, see also the Frequently Asked Questions page at our Technical Support Center: www.qiagen.com/FAQ/FAQList.aspx. The scientists in QIAGEN Technical Services are always happy to answer any questions you may have about either the information or protocols in this handbook (for contact information, visit support.qiagen.com).
Comments and suggestions
Unbalanced profiles, low signals
a) Incorrect volume of Fast Reaction Mix or Primer Mix
Check reaction setup and repeat amplification
b) Master mix not vortexed before distribution
Vortex master mix thoroughly and centrifuge briefly
Reduced peak heights of QS1 and/or QS2 in standard experiments
A slight peak height scattering of the Quality Sensors is usual and is not dependent on inhibitor influence.
During the validation, the analyst should evaluate the usual variation spectrum in relation to their certain sample types, and define a regular peak height range for both QS. A drop of the QS2 signal below 20% of the QS1 signal indicates inhibition of the PCR reaction.
Dominance of Quality Sensor peaks
QS1 and QS2 peaks are too dominant.
Using GeneMapper ID-X software choose under "Display Settings" a new setting for "all-dye range" to zoom in. The range should be between the QS1 and QS2.
Important: In addition, adjust in the "Analysis Method Editor" under "peak detector" the size-calling (Ranges; Sizing) to 75 450.
Sample preparation
Sample signal intensity must be increased.
Reduce the volume of the DNA Size Standard 24plex (BTO) to peak heights of about 500 RFU.
Purify the PCR products before starting the analysis. We recommend the MinElute® PCR Purification Kit (QIAGEN cat. nos. 28004 and 28006) for rapid and effective purification.
Matrix/spectral calibration is not appropriate
There are pull-up peaks between the dye panels (B, G, Y, R, P, O) with the current matrix/spectral calibration.
This matrix cannot be used for the analysis. Repeat the matrix generation/spectral calibration. Be sure to carefully follow the correct protocol for the specific analysis instrument.
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Comments and suggestions
Many peaks are labeled as off-ladder (OL) alleles in the samples
a) DNA Size Standard 24plex (BTO) was not defined or identified correctly.
Click the orange "Size Match Editor" icon in the upper toolbar of the GeneMapper ID or GeneMapper ID-X Software. Mark the orange fragments of all samples.
Always use DNA Size Standard 24plex for Investigator Human Identification PCR Kits.
b) Signal intensities are too high. If the peak heights of the samples are outside the linear detection range using Applied Biosystems 3500/3500xL Genetic Analyzers, the occurrence of stutters, split peaks, and artifacts may increase.
Reduce the injection time in increments to a minimum of 1 s, reduce the amount of the PCR amplification product for analysis, or reduce the quantity of DNA for PCR.
c) Bubbles in the capillary lead to pull-up peaks in all color panels ("spikes") that result in allele misnomer.
Repeat electrophoresis to confirm results. Check the maximum number of injections recommended by the instrument manufacturer. Setup a new capillary array, if necessary.
d) Differences in the run performance among the capillaries of a multicapillary analyzer may result in allelic assignment shift.
For reliable allelic assignment on multi-capillary analyzers, a number of allelic ladders should be run.
e) Low room temperature or low CE buffer temperature may result in fragment migration shifts or OL peaks.
Ensure ambient conditions are maintained as recommended by the instrument manufacturer. Ensure buffers are equilibrated to ambient conditions. Preheating of the CE instrument (~30 min) is recommended by the instrument manufacturer.
Injection/file of the allelic ladder is not appropriate
a) An additional signal can be identified as peak of the allelic ladder because of dysfunctions during the electrophoresis. If peaks of the allelic ladder are miscalled, the ladder cannot be used for the analysis.
Use a different injection/file of the allelic ladder and check the data of the analyzed sizes from the Size Standard (in bp) of the allelic ladder.
Always use the DNA Size Standard 24plex for Investigator Human Identification PCR Kits.
b) One peak of the allelic ladder is below the peak detection value (50200 RFU) of the analysis method used, and thus is not identified.
The allelic ladder must be loaded onto the analysis instrument at a higher concentration than the samples to be analyzed.
Alternatively, the allelic ladder data can be analyzed with a lower peak detection value in the analysis software.
c) One peak of the allelic ladder is not identified because it is outside the expected size range of the software (in bp).
Compare the length of the fragments (in bp) of the first allele in one color of the allelic ladder with the corresponding value in the categories. Then compare it with the other alleles.
d) Point alleles are not found.
Point alleles are alleles with at least 1 bp difference to the next integer allele. Check the settings of the analysis method. Lower the Peak Window Size value to 11 points.
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References
1. Bär, W. et al. (1997). DNA recommendations: Further report of the DNA Commission of the ISFH regarding the use of short tandem repeat systems. Int. J. Leg. Med. 110, 175176.
2. Szibor, R. et al. (2003). Cell line DNA typing in forensic genetics the necessity of reliable standards. Forensic Sci. Int. 138, 3743.
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Appendix A: Interpretation of Results
Post-PCR analysis and automatic allele assignment with suitable analysis software ensure a precise and reliable discrimination of alleles.
General procedure for the analysis 1. Check the DNA size standard. 2. Check the allelic ladder. 3. Check the positive and negative controls. 4. Analyze and interpret the sample data.
Pull-up peaks
Pull-up peaks may occur if peak heights are outside the linear detection range (see "Troubleshooting Guide"), or if an incorrect matrix was applied. They appear at positions of specific peaks in other color channels, typically with lower signal intensities. To prevent pull-up peaks, peak heights should not exceed thresholds.
Stutter peaks
The occurrence of stutter peaks depends on the sequence of the repeat structure and the number of alleles. The n 4 peaks are caused by a loss of a repeat unit during amplification of tetranucleotide STR motifs, caused by slippage effects of the Taq DNA Polymerase. The n 3 peaks appear during amplification of the trinucleotide STR motif D22S1045. These peaks should be interpreted using the Investigator Template Files for GeneMapper ID-X Software.
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Template-independent addition of nucleotides Because of its terminal transferase activity, the Taq DNA Polymerase may cause incomplete
adenylation at the 3'-end of the amplified DNA fragments. The artifact peak is one base shorter than expected (1 peaks). All primers included in the Investigator 24plex QS Kit are designed to minimize these artifacts. The peak height of the artifact correlates with the amount of DNA. Laboratories should define their own limits for analysis of the peaks.
Artifacts
Room temperature may influence the performance of PCR products on multi-capillary instruments, so shoulder peaks or split peaks might occur. If shoulder or split peaks appear, we recommend injecting the sample again. Ensure ambient conditions are maintained as recommended by the instrument manufacturer. Ensure buffers are equilibrated to the ambient conditions.
Appendix B: Varying PCR Volumes Using Investigator 24plex QS Kit
The Investigator 24plex QS Kit can be run with half reaction mix volumes (Fast Reaction Mix + Primer Mix). Note that while we have successfully tested the reduced reaction volume stated here, the highest overall success rates still have to be expected when using the full reaction volumes as recommended in the kit manual.
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Ordering Information
Product
Contents
Investigator 24plex QS Kit (100)
Primer Mix, Fast Reaction Mix 2.0, Control DNA, allelic ladder 24plex, DNA size standard 24plex (BTO), and nuclease-free water
Investigator 24plex QS Kit (400)
Primer Mix, Fast Reaction Mix 2.0, Control DNA, allelic ladder 24plex, DNA size standard 24plex (BTO), and nuclease-free water
Related products
Matrix Standard BT6 (50)
Matrix standard for 6-FAM, BTG, BTY, BTR2, BTP, and BTO, for Applied Biosystems 3500 Genetic Analyzers
Investigator Human Identification PCR Kits
Investigator Quantiplex Pro Kit (200)
For use on Applied Biosystems 7500 Real-Time Systems: Quantiplex Pro Reaction Mix, Quantiplex Pro Primer Mix, Quantiplex Pro Control DNA M1, QuantiTect Nucleic Acid Dilution Buffer
Cat. no. 382415 382417
386224
387216
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Product
Contents
Investigator Quantiplex Pro RGQ Kit (200)
For use on QIAGEN Rotor-Gene Q Real-Time Systems: Quantiplex Pro RGQ Reaction Mix, Quantiplex Pro RGQ Primer Mix, Male Control DNA M1, QuantiTect Nucleic Acid Dilution Buffer
Investigator 24plex GO! Kit (200)*
Primer Mix, Fast Reaction Mix 2.0, Control DNA, allelic ladder 24plex, DNA size standard 24plex (BTO)
* Larger kit sizes are available; please inquire.
Cat. no. 387316
382426
For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.
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Document Revision History
Date R7 11/2018 R8 12/2020 R9 02/2021
Changes
Added Appendix B
Added optional final extension. Updated the Ordering information section.
Table 3 is split into Tables 3a and 3b. Added "VeritiTM 96-Well Thermal Cycler" and "ProFlexTM 96-well PCR System" in the "Equipment and Reagents to Be Supplied by User" section.
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Notes
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Limited License Agreement for Investigator 24plex Kit Use of this product signifies the agreement of any purchaser or user of the product to the following terms: 1. The product may be used solely in accordance with the protocols provided with the product and this handbook and for use with components contained in the kit
only. QIAGEN grants no license under any of its intellectual property to use or incorporate the enclosed components of this kit with any components not included within this kit except as described in the protocols provided with the product, this handbook, and additional protocols available at www.qiagen.com. Some of these additional protocols have been provided by QIAGEN users for QIAGEN users. These protocols have not been thoroughly tested or optimized by QIAGEN. QIAGEN neither guarantees them nor warrants that they do not infringe the rights of third-parties. 2. Other than expressly stated licenses, QIAGEN makes no warranty that this kit and/or its use(s) do not infringe the rights of third-parties. 3. This kit and its components are licensed for one-time use and may not be reused, refurbished, or resold. 4. QIAGEN specifically disclaims any other licenses, expressed or implied other than those expressly stated. 5. The purchaser and user of the kit agree not to take or permit anyone else to take any steps that could lead to or facilitate any acts prohibited above. QIAGEN may enforce the prohibitions of this Limited License Agreement in any Court, and shall recover all its investigative and Court costs, including attorney fees, in any action to enforce this Limited License Agreement or any of its intellectual property rights relating to the kit and/or its components. For updated license terms, see www.qiagen.com.
Trademarks: QIAGEN®, QIAamplifier®, Investigator®, MinElute®, Rotor-Gene® (QIAGEN Group); Bio-Rad® (Bio-Rad Laboratories, Inc.); Eppendorf®, Mastercycler® (Eppendorf AG); 3500TM, Applied Biosystems®, 6-FAMTM, GeneAmp®, GeneMapper®, Hi-DiTM, POP-4®, ProFlexTM, VeritiTM (Thermo Fisher Scientific or its subsidiaries); GenBank® (US Department of Health and Human Services). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law.
02/2021 HB-1860-009 © 2021 QIAGEN, all rights reserved.
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