Assay quality
Various features such as single nucleotide polymorphisms (SNP) and secondary structures in primer annealing sites, are known to have a negative effect on amplification efficiency (see "Evaluation of the impact of single nucleotide polymorphisms and primer mismatches on quantitative PCR" by Boyle et al - 2009 - "Rapid detection of VHL exon deletions using real-time quantitative PCR" by Hoebeeck et al. - 2005 and "Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays" by Lefever et al. - 2013 ). Exclusion of these features from primer annealing sites is essential, since these can hamper proper hybridization of a primer to its target sequence. This may result in allelic drop out and reduced amplification. In addition, assay aspecificity titrates essential components of the PCR mix (e.g. dNTPs) away from the target of interest, resulting in an underrepresentation of the specific amplicon in the end product and potentially leading to non-equimolar end concentrations across PCR reactions requiring normalization prior to library preparation and sequencing.
Our state-of-the-art primer design pipeline takes into account all this and tries to generate assays having as little of these features present in their primer annealing site. Over 94% of our primers are free of SNPs, while for the remaining assays (5.99%), the number of SNPs is limited to one in over 92% of the assays (Figure 1). In these latter cases, the SNPs are located outside the last 5 nucleotides (3’ end) in 85% of the assays to further minimize the effect on amplification efficiency. More information regarding the specificity and associated sequencing coverage of our assays can be found here.
Our robust, ready-to-use assays, together with a high-quality oligo synthesis process ensures our customers a maximal PCR success rate. This, combined with fast delivery times, the uniform PCR conditions of our assays and the avoidance of laborious normalization procedures, allows rapid variant validation resulting in the shortest turnaround times.