Robust SNP genotyping by multiplex PCR and arrayed primer extension

Abstract

Background: Arrayed primer extension (APEX) is a microarray-based rapid minisequencing methodology that may have utility in 'personalized medicine' applications that involve genetic diagnostics of single nucleotide polymorphisms (SNPs). However, to date there have been few reports that objectively evaluate the assay completion rate, call rate and accuracy of APEX. We have further developed robust assay design, chemistry and analysis methodologies, and have sought to determine how effective APEX is in comparison to leading 'gold-standard' genotyping platforms. Our methods have been tested against industry-leading technologies in two blinded experiments based on Coriell DNA samples and SNP genotype data from the International HapMap Project.

Results: In the first experiment, we genotyped 50 SNPs across the entire 270 HapMap Coriell DNA sample set. For each Coriell sample, DNA template was amplified in a total of 7 multiplex PCRs prior to genotyping. We obtained good results for 41 of the SNPs, with 99.8% genotype concordance with HapMap data, at an automated call rate of 94.9% (not including the 9 failed SNPs). In the second experiment, involving modifications to the initial DNA amplification so that a single 50-plex PCR could be achieved, genotyping of the same 50 SNPs across each of 49 randomly chosen Coriell DNA samples allowed extremely robust 50-plex genotyping from as little as 5 ng of DNA, with 100% assay completion rate, 100% call rate and >99.9% accuracy.

Conclusion: We have shown our methods to be effective for robust multiplex SNP genotyping using APEX, with 100% call rate and >99.9% accuracy. We believe that such methodology may be useful in future point-of-care clinical diagnostic applications where accuracy and call rate are both paramount.

Figure 1

Multiplexing PCR and subsequent amplicon fragmentation results, prior to APEX reaction on HapMap Chip. (a) Standard multiplex PCR from a single Coriell DNA sample using optimally-designed primers [Additional files 1 &2] within seven unique multiplex groups (lanes 1–7; lane M shows 100 bp DNA ladder markers), showing wide range of amplicon sizes across the 50 SNP loci. (b) Purification, concentration and fragmentation of standard PCR amplicons. Lane 1 represents an aliquot of concentrated mixture of all seven multiplex products shown in Fig. 1a. Lane 2 shows the fragmentation result, generating single-stranded nucleic acid of 30–100 base length. (c) Multiplex PCR amplification of all 50 SNP loci in a single reaction tube using new PCR primer set [Additional file 6], showing 50-plex PCR products (individual SNP loci amplicons are unresolvable by agarose gel electrophoresis) from two Coriell DNA samples (lanes 1 & 2), plus a negative PCR control (lane 3). (d) Fragmentation of 50-plex PCR amplicons from aliquots of lane 1 & lane 2 samples shown in Fig. 1c.

Figure 2

HapMap Chip four colour microarray images showing successful de-multiplexing of 50-plex PCR from two Coriell DNA samples (a, b), plus a negative control sample (c), prior to image analysis and automated genotyping. The spots on the negative control image represent positive control probes [8, 14].

Figure 3

Simple scatter plots for SNP rs12466929 (A/G) from 50-plex data set (this SNP is representative of the entire set of 50 HapMap SNPs). For each plot the x-axis represents signal values for X allele (A for this SNP) and the y-axis represents signal values for Y allele (G for this SNP). All values are in log scale. Magenta, green, blue and black coloured symbols denote the classes YY (GG), YX (AG), XX (AA) and NN (negative control samples), respectively. Plot (1) combines the two ASO-APEX Left probes (one for each allele); plot (2) combines the two ASO-APEX Right probes (one for each allele); plot (3) is for the APEX Left probe; plot (4) is for the APEX Right probe. All the classifiers except APEX Left (plot 3) give well separated genotype clusters for this SNP. Dynamic variable selection is able to automatically weight these LDA classifiers in such a way that the homozygous AA cluster in plot (3) (blue) is able to contribute to the final call for such genotypes, even though AG (green) and GG (magenta) genotype clusters overlap somewhat for this Left APEX probe. Additional file 11 shows four-panel scatter plots for all 50 SNPs from the 50-plex data set.