We have developed a systematic, single-tube assay approach to reduce the chance of SNP genotyping error due to otherwise unidentifiable allelic drop-out during PCR amplification. Allelic drop-out in such cases would normally be caused by additional and rare genetic variation within the PCR primer site sequence itself. Our method is novel in that it does not require prior knowledge of the additional “hidden” genetic variation. The method has been tested in multiplex using a microarray-based SNP genotyping chip and results in the rescue of previously reported false genotype calls.

Introduction

In response to the ever-increasing numbers of research studies and diagnostic applications that involve the genotyping of single nucleotide polymorphisms (SNPs), the causes and consequences of genotyping errors have been the subject of several interesting publications in recent years (1,2). Solutions to detect and/or minimize errors have been proposed and instigated in the high-throughput research realm, and usually involve careful research study design and sophisticated statistical methods (2,3). In the lower-throughput research and diagnostic fields, genotyping errors have often been detected only by chance, and individual molecular assays have subsequently been redesigned to correct for the specific error-causing issue (4,5). One of the major causes of genotyping error is the presence of additional sequence variation close to the genetic marker that is actually under interrogation (1,6,7,8). This can cause partial or full allelic drop-out, due to inefficient primer hybridization in instances where the additional variation overlaps the primer sequence. It has been estimated that primer-site SNPs (primerSNPs) may account for half of all missed heterozygotes in some data sets, plausibly due to the missed heterozygotes also being heterozygous at a primerSNP (8). If the primerSNP is within a PCR priming site, allelic drop-out would be caused by preferential amplification of the chromosome with the perfectly matched allele, thereby leading the investigator to erroneously believe the locus being queried is homozygous.

Our laboratory has previously reported three such examples of allelic drop-out due to PCR primerSNPs (9) or primer “single nucleotide variants” in a study designed to validate robust microarray-based SNP genotyping methodology and analysis algorithms: 50-plex PCR was used to amplify 50 HapMap-characterized (www.hapmap.org) SNP loci from each of 49 human Coriell DNA samples (Coriell Institute for Medical Research, Camden, NJ, USA), followed by de-multiplexing and genotyping on mini-sequencing chips (9,10,11,12). We achieved a 100% genotype call rate and >99.9% accuracy with just three discordant genotypes. For each of these discordant genotype cases we identified additional single nucleotide variant sites that coincided with the positions delimited by the PCR primer sequences used for the 50-plex reaction, therefore consistent with full or partial allelic drop-out during the PCR. Only one of these sites was identified as an existing SNP (rs6871885), referenced in publicly available databases. As we previously concluded, more stringent due diligence at the PCR primer design stage would have avoided the deleterious effect of this primerSNP. Nevertheless, our identification of two hitherto unreported single nucleotide variants strengthens the concept that previously unknown variants cannot be avoided (8,13) and, as such, genotyping will always be somewhat at risk of error due to allelic drop-out. Solutions to these types of challenges have been proposed and tested, including independent sequencing from more than one amplicon which has been shown to dramatically reduce the impact of unknown primerSNPs on the rate of missed heterozygotes (8). We report here another, more systematic, single-tube multiplex PCR assay solution that we believe is a novel and very simple method to reduce the risk of otherwise unidentifiable allelic drop-out.

Materials and Methods

Original Multiplex PCR Methodology and Genotype Error Detection

Our original 50-plex PCR methodology with subsequent SNP genotyping assay is detailed in Podder et al. (9). Briefly, PCR primers were designed for 50 SNP loci, with amplicon sizes restricted to 100–200 bp. Each PCR primer had a common linker sequence designed at its 5′ end (5′-TACGACTCACTTAGGGAG-3′ for each of the left hand PCR primers, and 5′-CGATGTAGGTGACACTAG-3′ for each of the right hand PCR primers). The linkers serve to stabilize the PCR reaction, especially in multiplex mode. Specific PCR cycling conditions were adopted from a previously published study by Wang et al. (14). PCR and arrayed primer extension (APEX) genotyping assays were performed, and microarray image data were imported into SNP Chart genotyping software (SNP Chart v3.1, University of British Columbia, Vancouver, Canada) (11) and analyzed as described previously (9,15). Genotypes were compared with HapMap data for concordance. The SNP loci from the three discrepant genotype cases were independently re-amplified using longer-range PCR primers, followed by sequencing as previously described (9).