Quantifiler™ Trio DNA Quantification Kit - FAQs

How does the quantification solution provided by the Quantifiler Trio and HP DNA Quantification Kits and HID Real-Time PCR Software easily integrate with robotics for an automated workflow?

Robotic integration is enabled by the automation-friendly bundling of the Quantifiler Trio and HP DNA Quantification kits combined with the HID Real-Time PCR Analysis Software, which has been designed to facilitate automation with commonly used robotic platforms. In addition, our Human Identification Professional Services (HPS) team has extensive expertise in developing customized robotic scripts and conducting on-site verification and validation testing to help you achieve optimal performance with a wide range of platforms. The HPS team has already developed scripts for the Quantifiler Trio and HP DNA Quantification kits on several of the most common robotic platforms, and would be happy to consult with your lab on how to efficiently implement these solutions into your workflow. Please contact your local human identification (HID) account manager to learn more.

Can I use the basic SDS v1.2.3 software to analyze data generated with the Quantifiler Trio and HP DNA Quantification kits?

We do not recommend using the basic SDS v1.2.3 software. The Quantifiler Trio and HP DNA Quantification kits were developed and validated using the HID Real-Time PCR Software. The software ensures the optimal performance of new Quantifiler Trio and HP DNA Quantification kit-specific functionality, such as the automatic calculation of M:F ratios, Degradation Index and streamlined setup for the latest STR analysis kits including the Identifiler Plus, NGM Select and GlobalFiler kits.

What instrument can I use with the Quantifiler Trio and HP DNA Quantification kits and HID Real-Time PCR Software quantification solution?

The Quantifiler Trio and HP DNA Quantification kits and HID Real-Time PCR Analysis Software v1.3 have been validated to perform on the Applied Biosystems 7500 Real-Time PCR System.*

HID Real-Time PCR Analysis Software v1.4 has been validated to perform on the Applied Biosystems QuantSudio 5 and 7500 Real-Time PCR Systems.

* When used for purposes other than human identification, the instruments cited are for Research Use Only. Not for use in diagnostic procedures.

Does the Quantifiler Trio DNA Quantification Kit assay perform well with male:female mixture samples?

We evaluated male:female DNA ratios up to 1:4,000 with 20 pg/µL male DNA and were able to accurately quantify the male and female DNA fractions. For more detailed information, you can refer to the Mixture Study in the Quantifiler HP and Trio DNA Quantification Kits user guide.

How is female DNA detected with the Quantifiler Trio and HP DNA Quantification kits? Is there a specific marker for females?

By subtracting the male DNA concentration from the human DNA concentration, the concentration of female DNA can be approximated.

Have there been any updates to the standard curve in the Quantifiler Trio and HP DNA Quantification kits compared to previously released Quantifiler kits?

Yes, the recommended dilution series for both the Quantifiler Trio and HP Kits has been reduced from 8 concentration points to 5 points. This improvement over the previous kits was made in response to requests for more informative points on the standard curve combined with higher sample throughput. This change allows for approximately 6% more sample processing per 96-well plate. (Note: an optional 100 ng /µL standard point can be added for processing higher-concentration samples. Consult the user guide for more information.)

Are there any improvements or differences in the DNA standard in the Quantifiler Trio and HP DNA Quantification kits relative to the DNA standard included with previously released Quantifiler kits?

Yes, we have improved the homogeneity and stability of the DNA standard in the Quantifiler Trio and HP DNA Quantification kits by using lower-molecular-weight pooled genomic male DNA at a lower concentration (100 ng/µL vs. 150 ng/µL for the Quantifiler Duo kit standard) as well as improved manufacturing QC processes. To increase stability of the prepared standard dilution series, we recommend storing the prepared standards in low-bind tubes–for example, Applied Biosystems Nonstick RNase-free Microfuge Tubes, 1.5 mL (Cat. No. AM12450). Diluted DNA quantification standards can be stored up to 2 weeks at 2-8°C.

Which human target in the Quantifiler Trio and HP DNA Quantification kits do I use to measure DNA quantification for downstream processing?

The small autosomal (SA) target is used as the primary target marker for quantification, as DNA degradation and inhibition may impact the quantification of the large autosomal (LA) target.

Why are there two human quantification targets in the Quantifiler Trio DNA Quantification Kit and Quantifiler HP DNA Quantification Kit?

Both the Quantifiler HP and Trio kits have been designed with two human autosomal targets: a small autosomal (SA) target and large autosomal (LA) target. Both targets provide quantitative results for a sample. However, with increasing degradation, longer-target amplicons tend to decrease disproportionately relative to shorter-target amplicons. The relative amounts of LA amplicons and SA amplicons assist users in identifying possible sample degradation. This result is referred to as the degradation index.

How do improvements in the Quantifiler Trio DNA Quantification Kit, Quantifiler HP DNA Quantification Kit and HID Real-Time PCR Software correlate with Applied Biosystems STR kits?

The increased robustness (sensitivity, inhibition detection, and M:F ratio) of the Quantifiler HP and Trio chemistries was primarily designed to better correlate with downstream results when used in conjunction with the highly robust Applied Biosystems STR kits. These include Identifiler Plus, NGM SElect, Yfiler Plus, and GlobalFiler kits. The Quantifiler Human, Male, and Duo DNA Quantification Kits correlate more closely with the legacy Identifiler, Profiler, COfiler, SGMPlus, and Yfiler kits in terms of inhibition detection but do not offer any information on DNA degradation. The HID Real-Time PCR Software supports the setup and analysis of all currently available Applied Biosystems STR kits and Quantifiler chemistries.

How has the sensitivity of the Quantifiler Trio DNA Quantification Kit and Quantifiler HP DNA Quantification Kit improved compared to previous Quantifiler kits?

Internal validation data have shown that the Quantifiler HP and Trio kits can detect at the limit of ~1/10 of a cell equivalent (<1 pg/µL), which is a significant (>6x) increase in sensitivity compared to previous-generation Quantifiler kits. This is achieved through the use of multicopy target loci that provide much greater detection sensitivity than single-copy assay targets. The targets in the Quantifiler HP and Trio kits have been selected to maximize quantification sensitivity for challenging, low-concentration samples, including an improved male target (in the Trio kit) to deliver more accurate results for samples containing low concentrations of male DNA in the presence of high amounts of female DNA.

In addition, these multicopy target loci were carefully selected to minimize copy number variation between individuals and population groups, through the use of robust bioinformatics tools and extensive R&D testing. The improved sensitivity at sub-picogram levels of DNA, combined with high reproducibility, enables laboratories to more accurately and confidently identify and screen out samples that may not contain DNA sufficient for STR and/or Y-STR analysis–saving valuable time and resources in the lab.

What new features of the Quantifiler Trio and HP Quantification Kits and HID Real-Time PCR Software enable increased workflow efficiency?

Each new Quantifiler System includes the following:

• Updated chemistry
• Updated protocols for flexible sample processing
• A reduction in standard curve points, enabling more samples to be processed per run
• Faster time-to-results with a 33% reduction in cycling times
• Simple and streamlined instrument set up
• Simple and streamlined data analysis
• Sample normalization and STR set-up tables

How do the new Quantifiler HP DNA Quantification Kit and Quantifiler Trio DNA Quantification Kit deliver highly informative results in less time for challenging casework samples?

With an approximate cycling time of only 60 minutes, these new kits are ~33% faster than previous-generation Quantifiler chemistry–and they can provide more information regarding sample quantity and quality in a single reaction. The new quality index provides a more comprehensive view of sample quality (both degradation and inhibition) to facilitate up-front decisions on how to proceed to the STR reaction for optimal data recovery. Also, improved sensitivity at sub-picogram levels of DNA enables more accurate identification of samples that contain insufficient DNA to proceed with STR and/or Y-STR analysis, saving valuable time and resources in the lab.

What is a quality index and why is it important when using the Quantifiler DNA quantification kits?

The quality index is a measurement of both sample degradation (degradation index) and inhibition (IPC). A degradation index refers to the data observed when a sample displays a decrease in the measured amount of large DNA fragments compared to small DNA fragments. In the Quantifiler HP and Trio kits, the degradation index is measured by comparing the quantification results for the small autosomal (SA) and large autosomal (LA) targets and is used as a general indicator of whether large DNA fragments may perform poorly relative to small DNA fragments in STR reactions.

Forensic casework samples commonly contain inhibitors that can interfere with PCR amplification. The Quantifiler HP and Trio kits have been designed with an inhibitor tolerance that is ~5X higher than that of the Quantifiler Duo DNA Quantification Kit, which more closely correlates with highly robust Applied Biosystems STR chemistries such as those in the Identifiler Plus, NGM SElect, and GlobalFiler PCR Amplification Kits.* The degradation index can be affected by both the degree of degradation of the LA target and the presence of PCR inhibitors, so it's important to evaluate the degradation index in conjunction with the Internal PCR Control (IPC) results in the HID Real-Time PCR Software.

Taken together, the quality index helps to determine the overall sample quality and allows the lab an opportunity to select the optimal approach for each sample to maximize data recovery during STR analysis.*

*For Forensic or Paternity Use Only.

Our lab is currently using the Quantifiler Duo DNA Quantification Kit. What does the "Trio" in the Quantifiler Trio DNA Quantification Kit name signify?

There is an additional large autosomal (LA) target that has been added as part of the degradation index feature in the Quantifiler Trio DNA Quantification kit. This marker plus the improved small autosomal (SA) and male targets make three different quantification targets in the Quantifiler Trio kit, whereas the Quantifiler Duo kit has only two targets: one autosomal and one male. The new degradation index functionality in the Quantifiler Trio kit enables a more comprehensive view of sample quality (both degradation and inhibition), and the kit also has increased sensitivity and robustness to better correlate with more recently released Applied Biosystems STR chemistries, such as those in the Identifiler Plus, NGM SElect, and GlobalFiler PCR Amplification Kits.

Are the Quantifiler Trio DNA Quantification Kit and Quantifiler HP DNA Quantification Kit sold in different package sizes?

Both kits are available in a 400-reaction size. We have enhanced our kit packaging to help laboratories minimize the potential for contamination and facilitate automation capabilities. Instead of one bottle containing the reaction mix, we now have four 100-reaction mix tubes with sufficient overfill, which makes it easier to use one tube per 96-well quantification plate.

We have also packaged the DNA standard in a separate box. The boxes for both the reaction mix and the DNA standard are housed together with an outer sleeve that has peel-and-stick barcode labels, similar to those on GlobalFiler kits. This makes it easier to track reagent lot numbers and to separate reagents for storage upon arrival at the lab.

Are the Quantifiler Trio DNA Quantification Kit and Quantifiler HP DNA Quantification Kit available for sale in all regions?

Yes, both the Quantifiler Trio DNA Quantification Kit and Quantifiler HP DNA Quantification Kit are available globally.