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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.


Related in: MedlinePlus

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.
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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.

Mentions: We selected 50 SNPs from the HapMap database that had been previously genotyped and analyzed as part of the third quality control exercise on Illumina and Perlegen platforms, arguably the most accurate and best validated high-throughput methodologies for SNP genotyping to date. The randomly selected SNPs were located across multiple chromosomes and are listed in Additional file 1 online, along with details of the APEX probe sequences and PCR primer sequences. The genotyping arrays that are currently being developed and tested in our laboratory incorporate multiple redundant measures consisting of sense and antisense DNA-strand APEX probes plus allele-specific oligonucleotide (ASO) APEX probes for a total of six different probes per SNP [14], with each replicated five times on the array grid, which allows for more robust statistical averaging. Optimal PCR primer pairs were designed for each of the 50 SNP loci [Additional file 1] and seven multiplex PCR groups were set up that, together, would amplify all 50 loci [Additional file 2]. We obtained a set of 287 DNA samples from McGill University and Génome Québec Innovation Centre (one of the HapMap Project's genotyping centers). This set comprised 270 DNA samples from the Coriell Institute for Medical Research [22] plus hidden duplicates and negative controls, all of which our laboratory was blinded to. PCR [Fig. 1a and Fig. 1b] and APEX assays were performed on each of the samples, plus a 10% repeat set which was randomly selected by us to allow internal quality control and an initial assessment of genotyping concordance.


Robust SNP genotyping by multiplex PCR and arrayed primer extension.

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

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

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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.
Mentions: We selected 50 SNPs from the HapMap database that had been previously genotyped and analyzed as part of the third quality control exercise on Illumina and Perlegen platforms, arguably the most accurate and best validated high-throughput methodologies for SNP genotyping to date. The randomly selected SNPs were located across multiple chromosomes and are listed in Additional file 1 online, along with details of the APEX probe sequences and PCR primer sequences. The genotyping arrays that are currently being developed and tested in our laboratory incorporate multiple redundant measures consisting of sense and antisense DNA-strand APEX probes plus allele-specific oligonucleotide (ASO) APEX probes for a total of six different probes per SNP [14], with each replicated five times on the array grid, which allows for more robust statistical averaging. Optimal PCR primer pairs were designed for each of the 50 SNP loci [Additional file 1] and seven multiplex PCR groups were set up that, together, would amplify all 50 loci [Additional file 2]. We obtained a set of 287 DNA samples from McGill University and Génome Québec Innovation Centre (one of the HapMap Project's genotyping centers). This set comprised 270 DNA samples from the Coriell Institute for Medical Research [22] plus hidden duplicates and negative controls, all of which our laboratory was blinded to. PCR [Fig. 1a and Fig. 1b] and APEX assays were performed on each of the samples, plus a 10% repeat set which was randomly selected by us to allow internal quality control and an initial assessment of genotyping concordance.

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.


Related in: MedlinePlus