A High-Performing and Cost-Effective SNP Genotyping Method Using rhPCR and Universal Reporters

We have developed a novel dual enzyme chemistry called rhAmp SNP genotyping based on RNase H2-dependent PCR (rhPCR) that provides high signal and specificity for SNP analysis. rhAmp SNP genotyping combines a unique two-enzyme system with 3’ end blocked DNA-RNA hybrid primers to interrogate SNP loci. Activation of the blocked primers occurs upon hybridization to its perfectly matched target, which eliminates or greatly reduces primer dimers. A thermostable hot-start RNase H2 cleaves the primer immediately 5’ of the ribose sugar, releasing the blocking group and allowing primer extension. PCR specificity is further improved with the use of a mutant Taq DNA polymerase, resulting in improved allelic discrimination. Signal generation is obtained using a universal reporter system which requires only two reporter probes for any bi-allelic SNP. 1000 randomly selected SNPs were chosen to validate the 95% design rate of the design pipeline. A subsampling of 130 human SNP targets was tested and achieved a 98% call rate, and 99% call accuracy. rhAmp SNP genotyping assays are compatible with various qPCR instruments including QuantStudio 7 Flex, CFX384, IntelliQube, and Biomark HD. In comparison to TaqMan, rhAmp SNP genotyping assays show higher signal (Rn) and greater cluster separation, resulting in more reliable SNP genotyping performance. The rhAmp SNP genotyping solution is suited for high-throughput SNP genotyping applications in humans and plants.


Introduction
Genetic diversity at the single nucleotide polymorphism (SNP) level has been exploited in many branches of science and medicine. SNPs have been used to determine an individual's susceptibility to a variety of disease states ranging from breast cancer to cardiovascular diseases. Additionally, polymorphisms can be used to predict a drug's activity within a tissue. In pharmacogenetics, polymorphisms in genes encoding drug metabolism enzymes (DME) are used to help predict a drug's potency because the absorption, distribution, metabolism and excretion (ADME) of specific compounds can all be affected by genetic variations. Known polymorphisms in those genes are known as ADME SNPs. Additionally, SNPs are also commonly used as molecular markers in breeding and genetic research.
Multiple different chemistries or assays have been developed for SNP genotyping including mass spectrometry, oligonucleotide arrays, single stranded conformational polymorphism, and sequencing. Some of the most widely used technologies tend to be centered on a fluorescent based PCR assay (i.e. 5' nuclease, Molecular Beacons, Scorpion primers, KASP, and Invader) [1]. With all these methods, the allele-specific discrimination ability is solely based on a single factor, either probe hybridization or primer extension.
Here we present a new SNP genotyping method, rhAmp SNP, that combines the allele-specific hybridization and extension into a single assay. rhAmp SNP genotyping relies on two enzymes, RNase H2 (an endoribonuclease) and a mutant Taq DNA polymerase with enhanced allelic discrimination. The rhAmp SNP genotyping primers contain a single RNA base and are 3' end blocked. In addition, each of the allele-specific primers has a unique universal tail that incorporates sequence into the amplicon that is complementary to the reporter sequences. Rapid de-blocking of the primers by RNase H2 only occurs upon formation of a perfectly matched heteroduplex between the blocked RNA-containing primer and DNA target. Once de-blocked by RNase H2, the newly activated 3' end of the primer lays over the SNP site, and is interrogated by a mutant Taq DNA polymerase for allele-specific PCR. The major advantages of the rhAmp SNP genotyping chemistry include enhanced allelic discrimination by rhPCR, and high signal generation and cost reduction by the universal reporter system.  Table S2). DNA samples were quantified using a qPCR method targeting the RNase P (RPPH1) gene.

Assay Design
Universal reporter system: Of 10 million candidate sequences screened, a small set of universal primers and probes that met stringent thermodynamic properties (identical length, GC%, Tm, etc.) and had high sequence specificity against the human genome were selected. We further narrowed the probe set so that they did not form dimers and the pairwise edit distance was at least 4 for minimal interference among probes. A pair of the universal reporter probes were selected based on empirical testing (data not shown).
rhAmp SNP assay design pipeline: We have developed a sophisticated assay design pipeline that performs the following steps for each target SNP: 1) design all possible allele-specific primers (ASP), locus-specific primers (LSP) and their combinations; 2) evaluate the thermodynamic properties of the primers and amplicons, such as length, percent GC, Tm, sequence complexity, folding, etc.; 3) assess the impact of overlapping SNPs and repeats; 4) analyze the dimerization of assay primers, including self-and hetero-dimers; 5) evaluate primer compatibility, i.e. having similar Tm and length, particularly among ASPs; 6) determine the assay specificity against the human genome. Finally, based on the best combination of all the above parameters, an assay with the best overall quality is selected.

Description of rhAmp SNP Genotyping
The schematic describing rhAmp SNP genotyping is shown in Figure 1. Each rhAmp SNP assay is comprised of one locus-specific primer (LSP) and two tailed allele-specific primers (ASPs). Each primer has a single RNA base and a 3' end  The blocked primers are efficiently activated by the RNase H2 enzyme only in the presence of a perfectly matched RNA-DNA heteroduplex. Once the RNase H2 cleaves the primer, the 3' end is available for extension by the mutant Taq DNA polymerase (Figure 1(B)). Each ASP has a unique 5' tail sequence that becomes incorporated into the allele-specific amplicon. The tail region adds the necessary sequences for the binding of a universal primer and fluorescent probe (Figure 1(C)). The assays are designed such that the reference allele gives a signal in the FAM dye channel, and the alternative allele signal is detected in the VIC ® dye channel. The alternate allele fluorophore is Yakima Yellow ® , which has a very similar excitation and emission spectra as VIC dye. With such a similar excitation and emission profile, no spectral re-calibration is required.

Comparison of Wild-Type vs. Mutant Taq DNA Polymerase
While the RNase H2 enzyme exhibits significant differences in the rate of enzymatic cleavage of an RNA base in the presence or absence of a mismatch RNA-DNA heteroduplex [2], the rhAmp SNP assay also requires a DNA polymerase to complete allele-specific PCR. A novel mutant Taq DNA polymerase was developed to function effectively in the same buffer as the RNase H2 enzyme and provide improved allelic discrimination in comparison to the wild-type Taq DNA polymerase. Figure 2 shows the performance difference in the allelic discrimination (AD) plot between two master mixes, one containing the wild type Taq DNA polymerase (Figure 2(A)) and the other with mutant Taq DNA polymerase ( Figure 2(B)), with an example assay designed to detect SNP rs2269829. A total of 46 different human gDNA samples were tested at 3 ng of gDNA input per reaction. With wild-type Taq DNA polymerase, the three genotype clusters can be automatically called, however, the cluster separation is relatively poor, likely due to ASP mispriming, and some of the no template control replicates drift from the origin. Replacing the Taq with the mutant version improves the separation, tightness, and angles of the genotype clusters.

Performance of rhAmp SNP Genotyping Assays
Of 1000 common human SNPs randomly selected from the public dbSNP data-     For the Biomark HD, the assay was tested with 23 Coriell gDNA samples from a subset of the 90 ADME SNP gDNA panel samples, each at 150 ng per inlet. As noted in the methods, the thermal mix protocol is modified to achieve maximum assay sensitivity and specificity. rhAmp SNP assays perform well on these four major SNP genotyping platforms, with similar cluster angles and concordant genotyping calls achieved across all samples tested.

TaqMan vs. rhAmp SNP Genotyping
A robust SNP genotype call requires relatively high fluorescent signal, large cluster angle separation and tight clusters. In a study of 18 target SNPs, rhAmp SNP assays generated, on average, at least two-fold higher signal than TaqMan for both alleles (Supplemental Figure S1). The allelic discrimination plot and raw fluorescence of an ADME SNP rs776746 assayed by rhAmp SNP and Taq-Man genotyping chemistries was compared ( Figure 6). The rhAmp SNP assay achieves higher signal for all samples (Figure 6(A)), and more uniform signal for both alleles in heterozygote samples ( Figure 6(B)), resulting in a heterozygote cluster angle closer to the ideal 45 degrees.

Discussion
SNP genotyping using target-specific fluorogenic probes or allele-specific primers, such as TaqMan [3] and KASP assays [4], are two commonly used methods in medicine and agriculture. A new method, rhAmp SNP genotyping, which is based on allele-specific rhPCR combined with a universal fluorogenic reporter system, was first reported by Broccanello et al. [5]. This new SNP genotyping method has several advantages over both TaqMan and KASP.
First, the rhAmp SNP genotyping chemistry is a dual enzyme system with improved specificity of the allele-specific PCR, resulting in better genotyping cluster angles and separation (Figure 2). Dobosy and his colleagues [2] compared specificity of allele-specific PCR using unmodified PCR primers to that of 3' blocked rhPCR primers. Of 12 mismatched combinations, the mid-range dis- fied primers and 10.9 for 3' blocked primers, suggesting that rhPCR is at least 5-times better in allelic discrimination than standard PCR [2]. Secondly, the rhAmp SNP genotyping allele-specific and locus-specific primers are inactive Figure 5. Compatibility of rhAmp SNP genotyping with various qPCR platforms. Allelic discrimination plots for a rhAmp SNP assay targeting human ADME SNP rs7668258 are displayed with genotyping auto-calls assigned to samples run on the QuantStudio 7 Flex (A), CFX384 (B), IntelliQube (C) and Biomark HD (D). Allele 1 and Allele 2 calls indicate homozygous genotypes. Al-lele1/Allele2 call indicates heterozygous genotype. Figure 6. Comparison of rhAmp and TaqMan SNP genotyping. Assays targeting human ADME SNP rs776746 in the CYP3A5 gene were tested with 3 ng gDNA from 90 individuals. Allelic discrimination plot for all samples show higher signal and better cluster separation for the rhAmp assay (A) and multicomponent amplification curves for 5 samples with heterozygote genotype show higher and more uniform signal for the rhAmp SNP assay (B).
initially due the 3' end blocking group. Only upon hybridization to its perfectly matched target is the 3' blocking group readily removed by RNase H2 cleavage.
Therefore, primer dimers are eliminated or significantly reduced in the absence of target DNA (data not shown). High assay design rate (Table 1) is achieved in part due to relaxed rules checking primer to primer interactions. Thirdly, the rhAmp SNP genotyping method eliminates or reduces non-specific signal from universal reporters often caused by primer dimers. Some KASP assays can generate false genotyping clusters in the absence of targets or NTC wells, often due to non-specific interactions [6]. Non-specific interactions between Taq-Man probes and PCR primers also cause higher NTC signal in some TaqMan  [7]. Next, reduced primer dimers and the use of universal reporters with the rhAmp SNP genotyping system make multiplex SNP genotyping possible. Simultaneous detection of two SNPs in a single PCR reaction will reduce the assay cost and increase the throughput (unpublished data). Finally, use of the universal reporter system for any SNP or any species not only generates high fluorescent signals ( Figure 6) but also makes SNP genotyping cost-effective.

Conclusion
In conclusion, we have successfully applied a new rhPCR method for SNP genotyping from purified gDNA on existing qPCR instruments such as the Qua-ntStudio 7 Flex, CFX384, IntelliQube, and Biomark HD. This method provides a high-performance and cost-effective SNP genotyping solution for biomedical and agricultural applications. Figure S1. Signal-to-noise comparison between rhAmp and TaqMan SNP genotyping. Assays targeting 18 human SNPs were tested with 3 ng of Coriell gDNA from 46 individuals in 5 µL reactions with post-PCR read data collected and analyzed on the QuantStudio 7 Flex Real-Time PCR System software (Thermo Fisher). Signal to noise is determined for each allele by calculating the distance of the homozygous cluster from the no template control (NTC) (average assay NTC signal subtracted from the on-target allele signal from each homozygous sample). Compared to Taqman, rhAmp SNP genotyping results in higher cluster to NTC distance for both alleles in all 18 assays (A), with greater than 2-fold higher average cluster to NTC distance across the 18 assays (B).