Independent academic study compares 3 qPCR-based SNP genotyping methods in plants

New era in agriculture fueled by SNP genotyping

Single-nucleotide polymorphisms (SNPs) are powerful markers for monitoring the presence of critical qualitative and quantitative traits when breeding plants and animals of agricultural importance. The ubiquitous distribution of SNPs throughout a given genome and the economic importance of propagating specific traits in livestock and crops led to the development of several SNP detection technologies, including sequencing, array platforms, and quantitative polymerase chain reaction (qPCR). qPCR-based SNP genotyping has become the dominant method when small numbers of SNPs are to be detected in a large number of plant and animal samples. PCR-based methods are an affordable approach, providing accurate genotyping results using high-throughput processing with low sample input.

rhAmp SNP Genotyping System improves on PCR-based SNP genotyping methods

Two commonly used SNP genotyping platforms are TaqMan^® assays (Thermo Fisher) and Kompetitive Allele Specific PCR (KASP^® assays; LGC Genomics). TaqMan assays are based on real-time detection of fluorescently-tagged, allele-specific probes, while the KASP system uses end-point fluorescence to discriminate tagged alleles. Both platforms, however, can exhibit low signal, causing imperfect allelic discrimination, and have a relatively high cost of implementation.

IDT developed a novel genotyping method which addresses the weaknesses of these 2 platforms and provides a lower overall cost of SNP genotyping for end-users. The rhAmp SNP Genotyping System (IDT) is based on RNase H2-dependent PCR (rhPCR) combined with a universal reporter system. (For a description of how this unique 2-enzyme system and RNA-DNA hybrid primers precisely interrogate target SNPs, see the article, Better PCR genotyping—obtain greater precision with RNase H2 activation of assay primers.)

Independent study demonstrates exceptional performance of rhAmp SNP Genotyping Assays compared to other vendors’ assays

Dr Chiara Broccanello and her colleagues perform genotyping experiments in the Department of Agronomy, Food, Natural Resources, Animals, and Environment, at the University of Padova, Italy. In this study [1], they compared the accuracy, sensitivity, and cost of TaqMan, KASP, and rhAmp SNP genotyping platforms using 33 pre-validated SNPs in a set of 96 sugar beet (Beta vulgaris L.) individuals originating from 12 parental lines. They used quantitative metrics such as call rate, no template control (NTC) location, cluster to NTC distance, cluster angle separation, and cluster spread for comparison of the 3 methods (Figure 1).

Figure 1. rhAmp SNP Genotyping Assays provide superior allelic discrimination compared to TaqMan and KASP assays. Sugar beet (Beta vulgaris) samples (96 individuals from 12 parental lines) were genotyped for SNP103 using custom SNP assays (TaqMan, KASP, or rhAmp assays provided by Thermo Fisher, LGC Genomics, and IDT). The study examined an additional 32 pre-validated SNPs (with the 33rd shown here).  Allelic specificity was obtained using 2 probes labeled with FAM and VIC^® (Thermo Fisher) reporter dyes in TaqMan assays, FAM-HEX in KASP assays, and FAM-Yakima Yellow^® (YY; ELITech Group) in rhAmp assays. All reactions were performed in a 5 µl volume in 384-well plates, using the QuantStudio™ 12K Flex Real-Time PCR System (Thermo Fisher). ●, ● homozygote genotypes; ● heterozygote genotype; ■ No template control (NTC), x no call. Figure 1 by Broccanello et al. [1] is licensed under CC BY 4.0.

rhAmp SNP Genotyping Assays demonstrated 100% concordance with both TaqMan and KASP assays for 24 of the 33 SNPs, and >99% concordance for the remaining 9 SNPs examined. The call rate was consistently >97% across the 3 methods, with the highest values obtained using rhAmp assays (98.1%). The rhAmp SNP Genotyping Assays also showed a higher cluster to NTC distance and a lower cluster spread than either TaqMan or KASP methods.

Dr Broccanello and colleagues additionally compared assay sensitivity, the time required for preparation and data analysis, and cost per assay. The research team performed an analytical sensitivity test using various amounts of input DNA ranging from 0.1 to 100 ng per reaction. They evaluated the limit of detection (LOD) for 2 alleles of SNP103 using normalized fluorescent signal levels (ΔRn). Whereas KASP was unable to ascertain SNP states for DNA inputs <0.9 ng per reaction, both TaqMan and rhAmp assays were able to successfully call SNP genotype with as little as 0.2 ng of DNA per reaction (Table 1).

Data also demonstrated that rhAmp assays provide consistently higher signal levels compared to both TaqMan and KASP assays over a wide range of higher DNA inputs (10–100 ng; Figure 2). The strong signal provided by rhAmp assays can be particularly valuable with automated calling in high throughput applications. All 3 methods required similar setup and data analysis time; however, rhAmp assays offered the lowest cost per reaction in this study.

Figure 2. rhAmp assays consistently provide higher signal levels compared to both TaqMan and KASP assays. The LOD values for 2 SNP103 alleles were determined with TaqMan, KASP, and rhAmp assays using 10–100 ng input DNA. Figure 2 by Broccanello et al. [1] is licensed  under CC BY 4.0.

rhAmp assays take PCR-based SNP genotyping to the next level

Dr Broccanello and colleagues concluded that the rhAmp assays are an improvement in the efficiency of SNP detection compared to both the TaqMan and KASP methods, and that rhAmp SNP Genotyping Assays offer plant and animal breeders a better alternative for their molecular breeding practices.