Detecting and quantifying small percent copy number differences with a high degree of precision
Welcome to the third installment of our six part series to introduce you to digital PCR (dPCR). In this edition we’ll look at how dPCR is enabling researchers’ abilities to achieve precise copy number variation (CNV) analysis.
A range of methods for CNV detection is currently available, including fluorescent in situ hybridization (FISH), comparative genomic hybridization (CGH), array comparative genomic hybridization (aCGH), real-time PCR (qPCR), next-generation sequencing (NGS), and dPCR.
CNV is one of the most common genetic variations in the human genome
CNV is a modification in the genome where the number of copies of a genomic DNA sequence differs from a reference or standard. Genomic alterations such as insertions (as small as a few basepairs up to 10 kb), deletions (as small as a few basepairs but as large as several Mb), inversions, or translocations can lead to biallelic or multiallelic CNVs. CNVs are one of the most common genetic variations in the human genome and have been implicated in many diseases, including cancer and inherited disease susceptibility.
dPCR enables precise detection and accurate quantification of CNV
Despite advances in some of the aforementioned technologies, in many cases, measurements are not sufficiently precise for determining CNVs where the ratios between the target and reference are very small.
Currently real-time qPCR is one of the most commonly used technologies to validate CNV in populations, despite typically requiring many replicates to achieve the desired precision. More than 1.6 million predesigned TaqMan® Copy Number Assays and design tools are available to evaluate CNVs using real-time qPCR instruments and software. They offer simple workflows along with specific and highly reproducible copy number results and remain viable in cases with lower sensitivity level requirements, e.g. >1.1x precision, larger sample numbers, and general screening. TaqMan® Copy Number Assay workflows can be automated so that several hundred to thousands of samples can be processed in a single day.
dPCR is rapidly emerging as an accurate, precise, and simple method for analyzing CNV, one that enables the detection of low percent copy number differences to be detected and accurately quantified.
Performing CNV analyses using the QuantStudio™ 3D Digital PCR System
The QuantStudio™ 3D Digital PCR System a simple and affordable dPCR platform to enable precise and accurate absolute quantification for your nucleic acid targets at both low and high copy numbers, as well as high reproducibility and superior resolution compared to other techniques.
To demonstrate dPCR’s ability to detect higher copy number, a panel of nine gDNA samples was analyzed using the QuantStudio™ 3D and a TaqMan® Copy Number Assay for the CCL3L1 gene, which is a chemokine gene of variable copy number among individuals. Results indicated that the samples contained variations from zero to eight copies. (See A.)
A statistically measurable difference – less than a 1.2 fold – between samples containing seven and eight copies was clearly discernable due to the high degree of precision achieved using dPCR.
To further demonstrate dPCR’s ability to deliver highly precise measurements, two samples containing 2 copies and 3 copies of the CCL3L1 gene respectively, were mixed at different ratios to simulate samples with predicted copy numbers between two and three copies in 0.1 copy increments. (See B.)
Copy numbers for the mixed samples were measured using the QuantStudio™ 3D and plotted against predicted copy number. The results indicate the ability to resolve very small differences in copy number approximately <5% – simulating a heterogeneous cell population.
A B
CAPTION:
Measured copy number plotted against expected copy number for each sample. Blue dots – copy number from individual chips. Red crosses – mean of all measurements for the given sample. Grey bars – standard deviations. Dotted line – 99% confidence interval for a predicted response.
Hear what researchers are saying about performing CNV with dPCR
Additional examples of using dPCR to help achieve precise and accurate CNV analysis can be found at the following video links:
Ask TaqMan: Detecting Copy Number Variation (CNV) with Digital PCR
Copy Number Variation (in HER2) Using Digital PCR
Digital PCR Used to Assess CNV in HER2 with QuantStudio™ 3D
Digital PCR Measures Her-2 Copy Number in Breast Cancer Samples
Her-2 Amplification Assessment in Breast Cancer FFPE Samples
Also available is this short-and-to-the-point research paper: “Copy number variation in breast cancer translational research: The QuantStudio™ 3D Digital PCR System as a cost-effective and sensitive alternative for HER2 gene amplification assessment”
Finally, grab a brown bag lunch and log in to this informative on-demand webinar, Beyond the Limits with Digital PCR, with featured speaker Trish Hegerich, Senior Applications Scientist, Digital PCR Technology, Life Technologies. Lunch and learn about how digital PCR brings you beyond the limits of real-time PCR for CNV analysis.
Conclusion
Determination of CNV requires both precision and accuracy. Commonly used technologies such as real-time qPCR, while adequate, have their limitations, e.g., relative quantification, real-time data collection, no sample partitioning, and requiring a standard curve. All the more reason to discover the advantages of dPCR. The QuantStudio™ 3D, a simple and affordable dPCR platform, along with a wide range of TaqMan® CNV Assays, helps enable precise and accurate absolute quantification for your nucleic acid targets at both low and high copy numbers, as well as high reproducibility and superior resolution is within your reach.
Learn More
Learn more about how the QuantStudio™ 3D Digital PCR System is designed to simplify workflows that enable precise and accurate copy number results with minimal hands-on time and a fast turnaround time. And be sure to take a moment to watch our videos to see and hear how scientists worldwide are using QuantStudio 3D to perform CNV analysis in leading-edge cancer research
Read all of the Let’s Go Digital PCR Series:
Go Digital PCR: Pt 1 – Next-Gen Quantification
Go Digital PCR: Pt 2 – QuantStudio™ 3D
Go Digital PCR: Pt 3 – Copy Number Variation
Go Digital PCR: Pt 4 – NGS Libraries
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