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Robust SNP genotyping by multiplex PCR and arrayed primer extension.

Podder M, Ruan J, Tripp BW, Chu ZE, Tebbutt SJ - BMC Med Genomics (2008)

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St, Paul's Hospital, University of British Columbia, Vancouver, BC, V6Z 1Y6, Canada. mpodder@mrl.ubc.ca

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.

No MeSH data available.


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].
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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].

Mentions: PCR [Fig. 1c and Fig. 1d] and APEX assays [Fig. 2] were performed on each of the samples, including negative controls. Microarray image data were imported into SNP Chart and analyzed as described previously. Genotype calling was performed using three independent methods: 1. manual calling in SNP Chart; 2. auto-calling with MACGT; and 3. auto-calling by LDA using dynamic variable selection. Genotypes were compared to HapMap data for concordance. One SNP (rs7693776) was monomorphic (TT) across all samples genotyped. Results are presented in Table 2 and [Additional files 7, 8, 9, 10]. Manual genotype calling, although time-consuming and vulnerable to user-subjectivity issues [14,23], is nevertheless an accurate and validated way to interpret APEX data, especially at low spot intensity levels (see above). In addition, manual calling does not require the use of a training set. Of the 49 Coriell DNA samples (one sample out of the random set was a blinded negative control sample) assayed across 50 SNPs, manual calls were made for all possible 2,450 genotypes (100% assay completion and 100% call rate). Of these, 2,448 were concordant with HapMap data (99.92%). The two discrepant genotypes were for two different samples each at different SNP loci. Interestingly, the SNP Charts for these two genotypes showed high quality data, and the same samples/genotypes had previously been concordant with HapMap in the initial data set [Additional file 3, and discussed further below].


Robust SNP genotyping by multiplex PCR and arrayed primer extension.

Podder M, Ruan J, Tripp BW, Chu ZE, Tebbutt SJ - BMC Med Genomics (2008)

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].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2266772&req=5

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].
Mentions: PCR [Fig. 1c and Fig. 1d] and APEX assays [Fig. 2] were performed on each of the samples, including negative controls. Microarray image data were imported into SNP Chart and analyzed as described previously. Genotype calling was performed using three independent methods: 1. manual calling in SNP Chart; 2. auto-calling with MACGT; and 3. auto-calling by LDA using dynamic variable selection. Genotypes were compared to HapMap data for concordance. One SNP (rs7693776) was monomorphic (TT) across all samples genotyped. Results are presented in Table 2 and [Additional files 7, 8, 9, 10]. Manual genotype calling, although time-consuming and vulnerable to user-subjectivity issues [14,23], is nevertheless an accurate and validated way to interpret APEX data, especially at low spot intensity levels (see above). In addition, manual calling does not require the use of a training set. Of the 49 Coriell DNA samples (one sample out of the random set was a blinded negative control sample) assayed across 50 SNPs, manual calls were made for all possible 2,450 genotypes (100% assay completion and 100% call rate). Of these, 2,448 were concordant with HapMap data (99.92%). The two discrepant genotypes were for two different samples each at different SNP loci. Interestingly, the SNP Charts for these two genotypes showed high quality data, and the same samples/genotypes had previously been concordant with HapMap in the initial data set [Additional file 3, and discussed further below].

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: The James Hogg iCAPTURE Centre for Cardiovascular and Pulmonary Research, St, Paul's Hospital, University of British Columbia, Vancouver, BC, V6Z 1Y6, Canada. mpodder@mrl.ubc.ca

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.

No MeSH data available.