The next evolution of PCR for genotyping
A successful genotyping assay should deliver high call rate (% samples with genotype calls) and high accuracy (% samples with correct calls). Good cluster separation with low trailing data points and high signal-to-noise ratio are important features of the allelic discrimination plot (also known as a "cluster plot" or an "AD plot”).
IDT developed the rhAmp SNP Genotyping System to meet all of these criteria, enabling successful generation of high quality genotyping data. This novel technology not only offers a collection of predesigned assays for human SNPs, but also includes a custom assay design pipeline for newly discovered human SNPs or for assay designs for other species. (See the article, Better PCR genotyping—obtain greater precision with RNase H2 activation of assay primers, for an explanation of how this unique 2-enzyme system and RNA-DNA hybrid primers are used to precisely interrogate target SNPs.)
Millions of up-to-date, predesigned assays
The rhAmp SNP Genotyping System predesigned human assay library contains the largest number of commercially available assays—more than 11 million assays for human SNPs. These assays demonstrate consistently high performance, with >99.5% call accuracy in over 90% of assays tested (Figure 1). The library also includes a broad selection of functionally validated ADME SNP assays [i.e., assays that target SNPs in genes responsible for the absorption, distribution, metabolism, and excretion (ADME) of pharmaceutical compounds] that deliver 100% call accuracy.
Figure 1. More than 90% of rhAmp SNP Genotyping assays tested provide >90% call rate and >99.5% call accuracy. Genotyping was performed on 213 rhAmp SNP Genotyping System assays in 5 µL reactions, using human gDNA (3 ng) from 46 individuals (Coriell Institute). Analysis was conducted using QuantStudio™ 7 Flex Real-Time PCR System software (Thermo Fisher).
Design pipeline provides high quality assay design
The IDT proprietary design pipeline, an integral part of the rhAmp SNP Genotyping System, provides quality assay designs with a high design success rate per target tested. The following design pipeline parameters ensure the best assay performance.
- Robust thermodynamics: Each assay is reviewed for several thermodynamic properties. These include amplicon size, RNase H2 cleavage efficiency, primer length, GC content, melting temperature (Tm), sequence complexity, secondary structure folding, and primer dimer formation.
- Better assay placement: The effect of possible underlying SNPs and sequence repeats is evaluated to select the best assay positioning.
- High specificity: An algorithm checks for off-target amplification against a reference genome, in a simulated PCR. The algorithm also checks designs against alternative alleles to ensure assays are highly allele specific.
Better performing assays for difficult genomic regions
The rhAmp SNP Genotyping System provides high performing designs where other technologies often fail, such as genomic regions with thermodynamic issues or high GC content. Due to its unique technology, rhAmp assays can be used with amplicons as small as 40 bp. This feature enables quality assay designs in areas where other technologies with longer amplicon requirements are hindered by sequence complications. In addition, the design algorithm evaluates both strands for selecting allele-specific primers when genomic content near the target SNP is problematic.
The rhAmp SNP Genotyping Assays generate strong, uniform signal even in difficult genomic regions. The system uses universal reporter probes that are not target specific. These probes were selected from more than 10 million candidates to meet stringent thermodynamic properties such as identical length, GC content, and Tm. Since they are designed to recognize the common tail in the primer sequence, there is no need to design a new probe for each target, as is the case for other commercially available assays.
The rhAmp SNP Genotyping Assays use a novel 5’ nuclease assay chemistry as the detection method for the universal reporter system. As a result, the assays deliver an increased signal-to-noise ratio compared to traditional 5’-nuclease chemistry.
Taken together, these features enable rhAmp SNP Genotyping Assays to achieve superior performance for difficult genomic regions and challenging sequences.
Custom assay design tool for new and non-human SNPs
The rhAmp SNP Genotyping Design Tool can be used to generate custom assay designs using the same bioinformatics process employed to create the human predesigned assays (Figure 2). For newly discovered SNPs that are not part of the predesign database, the algorithm performs the extensive quality checks mentioned above to generate the best assay design. Non-human assays may also be created after providing high-confidence sequences. (Currently, genome QC is only available for human sequences).
Figure 2. Design tool enables custom designs for new and non-human assays.
Use the rhAmp SNP Genotyping System as a novel, cost-effective genotyping solution that provides superior allelic discrimination. This innovative, dual-enzyme system is equipped with a robust and flexible design pipeline to provide the best assay designs and ensure a high design success rate.
Learn more about the rhAmp SNP Genotyping System and assay design tool.