Pharmacogenomics Support—Getting Started

Pharmacogenomics (PGx) is the study of genetic variation as it relates to drug response. PGx studies involve testing individuals for multiple variants in drug metabolism enzyme (DME) and transporter genes. These studies are increasing in significance as personalized medicine becomes a reality in standard practice. We offer simple workflows for DME genotyping and copy number variation (CNV) analysis.

We have a wide variety of products. Please see the table below with links to the products that are commonly used:

*Accessory kits are included with all custom OpenArray® plate orders. The only exception is for products that are sold as individual OpenArray® plates, such as the PGx panel.

The OpenArray® instrument allows for multiple custom configurations, as shown in the table below:

Genotyping data is analyzed using TaqMan® Genotyper™ Software. Copy number variation is analyzed using CopyCaller™ Software. AlleleTyper™ Software accepts the results of both the TaqMan® Genotyper™ and CopyCaller™ Software and uses those results to assign the haplotype of the samples. The cloud-based AlleleTyper™Software is accessed from the Life Technologies™ website. You will be prompted to enter your lifetechnologies.com account info (or register for one if you do not have a Life Technologies™ account).

Yes, we have the TaqMan® OpenArray® PGx Panel, QuantStudio® 12K Flex, Cat. No. 4475395. This panel contains 158 assays, and you can view a list of the assays and the plate layout here: TaqMan® OpenArray® PGx Panel, QS, Plate Layout & Contents

For CYP allele nomenclature, see The Human Cytochrome P450 (CYP) Allele Nomenclature Database and PharmGKB, the Pharmacogemonics Knowledgebase. For troubleshooting the TaqMan® assays, see the Pharmacogenomics Experiments User Guide.

With our optimized MagMAX protocol and a suitable swab (such as the FLOQSwabs™ flocked swabs), we routinely see 70–200 ng/µL . As a result, preamplification should generally not be required. Initial troubleshooting should focus on getting good yields without preamplification. For information on preamplifying, please see this application note: A complete workflow for pharmacogenomics using the QuantStudio® 12K Flex Real-Time PCR System.

To perform a pre-read, Get Plate IDs, input the setup file either with Genotyping.edt or spf for your runs.
Check the box for Include Pre-read and uncheck Include Amplification.
Edit the file location and the file name and start the run.

After your run has ended, you can close it and perform other experiments.
If you wish to perform the post-read, put the OpenArray® plate in the same position it was in when you did the pre-read, and select Get Plate IDs.

Be sure to select the *.eds file that you generated for the pre-read as the setup file, and start the run. You cannot use the *.spf or *.edt file—it must be the *.eds file. If you place the array at the wrong position, the software will prompt you to place it in the correct position.

 All of the arrays are functionally tested. The array QC includes the following tests:
- ROX Presence Test: This is a visual fluorescent detection test to assess whether an assay mixture is loaded onto the OpenArray® Genotyping Plate. The ROX Presence Test is run on every TaqMan® OpenArray® Genotyping Plate.
- Assay Presence Test: For human assays, this is a functional test using human genomic DNA.
Cluster plots are reviewed for performance quality. For nonhuman assays, this is a visual fluorescent detection test to assess whether the TaqMan® Assays were loaded onto the OpenArray® Genotyping Plate at a detectable concentration. The Assay Presence Test is run on two TaqMan® OpenArray® Genotyping Plates from each manufacturing batch of 12 arrays.

You can email techsupport@thermofisher.com and submit a request. We will provide you with the necessary files within 48 hours. Please be prepared to provide the lot numbers for the array plates, as well as the sales order number.

The platform is CFR21 Part 11 enabled, but is currently not FDA-approved. The system is manufactured under ISO 13485. The system has audit-enabled software, sample tracking, and LIMS integration. We also offer IQ/OQ support and IPV and CSV. More information about these services is described here:

Installation Qualification (IQ)
The IQ procedures help ensure that instrumentation is delivered and installed according to our stringent specifications, which include documenting the shipment integrity, the system components, and compatibility with the system configuration.
We recommend that you have an IQ service performed at the following times:

• At the time of instrument installation
• When an instrument is moved to another location or laboratory
• After functionality is added to the system, such as a hardware or software upgrade
• After components are added to the system
• Before a previously installed system will be used in a regulated test environment for the first time (e.g., an instrument used for basic research transitions for use in clinical trials testing

Operation Qualification/Instrument Performance Verification (OQ/IPV)
The OQ/IPV service verifies and records the instrument’s ability to meet specified performance criteria after installation, repetitive use, relocation of the instrument, or major service events. The OQ/IPV service involves comprehensive testing of the complete system using established conditions and known sample characteristics.
A key benefit of the OQ/IPV is to help ensure the accuracy and precision of the instrument according to manufacturer’s specifications. Instruments should be regularly tested to confirm performance according to manufacturer’s specifications and identify possible problems due to normal wear or inadequate user maintenance.
We recommend that you have an OQ/IPV service performed at the following times:

• At instrument installation
• On a periodic basis in accordance with your standard operating procedures
• After a major repair
• After relocating an instrument to another laboratory
• After functionality is added to the system, such as a hardware or software upgrade
• After any software change that affects system security, data integrity, or administrative controls
• Before a previously installed system will be used in a regulated test environment for the first time (e.g., an instrument used for basic research transitions for use in clinical trials testing

Computer System Validation (CSV)

Our Computer System Validation consists of the following:

On-site standard validation performed by Life Technologies™ certified representatives. This consists of on-site testing and validation of typical system requirements to support your compliance, including:

• Comprehensive user requirements and associated test cases to verify that electronic records are generated, maintained, and archived in an accurate, reliable, and secure manner
• Validation methodology based on GAMP5 guidelines and risk-management practices
• Expedited delivery within two weeks following final document approval

Computer System Validation Service

• Applied Biosystems® trained and certified representatives
• Test and validate system requirements over multiple days to demonstrate compliance with GLP regulations and guidelines
• Completion of 12 documents and standard Computer System Validation (CSV) IQ/OQ/PQ test cases
• Industry-expert reviewed and approved CSV approach
• Multi-system pricing available
• Expedited delivery

Any solid tissue, blood, serum, buccal swabs, plasma, FFPE, or saliva samples can be used as the DNA source. Preamplification may be required for some of these sample types, depending on purity and concentration. The recommended amount of template for each single (through‐hole) reaction in a TaqMan® OpenArray® plate is 250 copies of the haploid genome, equivalent to 0.84 ng for human DNA samples.

Please note: The recommended starting concentration for human DNA samples is 50 ng/μL to obtain 250 copies of the haploid genome, per through‐hole.

We do not have standard control samples for the TaqMan® DME assays. However, you can review the test data for the genotyping and copy number assays to find Coriell controls to confirm results with our test data.

Alternatively, plasmids can be purchased for testing, or obtained as part of an analytical validation package.

You can download your SPF plate files from here.

We recommend using the TaqMan Genotyping Master Mix (Cat. No. 4371357). Please see the Pharmacogenomics Experiments User Guide Addedum for details on how much to order.

Additional OpenArray® plates can be run off-line using a flat-block thermal cycler. The two thermal cyclers we recommend are the GeneAmp® PCR System 9700 and the ProFlex™ PCR System. 

You need an OpenArray® Plate Adaptor, Cat. No. 4472306. You do not need a compression pad. This part number works with both the GeneAmp® PCR System 9700 and the ProFlex™ PCR System.

You need an OpenArray® Plate Adaptor, Cat. No. 4426824, and a compression pad, Cat. No. 442825.

• Position the adaptor.

1.     Insert the two adaptor pins into the circular slots on the module.

2.     Be sure the adaptor sits flat.

• Position the TaqMan® OpenArray® plates.

1.     Orient each plate with the barcode facing up and along the edge of the block, away from the center of the adaptor.

2.     Fill up the right sample block first when placing the adaptor on the module (right sample block is the primary control block).

• Fill up the center slots before any edge slots.

1.     (Recommended) Insert unused TaqMan® OpenArray® plates into any empty slots.

• Place the compression pad over the TaqMan® OpenArray® plates (only needed for NT-OpenArray® plates, not for QuantStudio™ 12K), with the Teflon coating (smooth side) facing upwards and away from the plates.
• Slide the heated cover over the compression pad, then pull down the handle.

For more detailed instructions, refer to the Dual Flat Block GeneAmp® PCR System 9700 User Guide. 

These instructions may be found on page 5 of the Dual Flat Block GeneAmp® PCR System 9700 User Guide.

93°C for 10 min, ramp rate 100%

50 cycles of: 
95°C for 45 sec, 84% ramp rate
94°C for 13 sec, 100% ramp rate
53°C for 2 min 14 sec, 44% ramp rate

25°C for 2 min, 100% ramp rate

Optional: Add a post-PCR overnight hold as shown below: 
4°C for 99 min 59 sec, 100% ramp rate

These instructions may be found on page 79 of the ProFlex™ PCR System User Guide.

Reaction volume: 33 nL
Cover temperature: 70°C

94°C for 600 sec, ramp rate 1.3

50 cycles of: 
99°C for 30 seconds, ramp rate 1.3
54°C for 21 seconds, ramp rate 1.1
57°C for 90 seconds, ramp rate 0.1

To perform pre-read and post-read, choose Get Plate IDs and input the setup file either with Genotyping.edt or .spf for your runs.

Check the box for Include Pre-read and uncheck Include Amplification.

Edit the file location and the file name and start the run.

After your pre-read run has ended, you can either keep the run console on to continue with the post-read, or you can close it and perform other pre-read runs.

If you closed the run console and wish to perform the post-read, put the OpenArray in the same position as it was when you did the pre-read and choose Get Plate IDs again. Select the eds file that you had performed pre-read as the setup file and start the run. Note: it is important here that you select the .eds file, NOT the .spf or .edt.

If you have placed the array at the wrong position, the software will prompt you to select the correct position.