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Analysis of copy number variation using quantitative interspecies competitive PCR.

Williams NM, Williams H, Majounie E, Norton N, Glaser B, Morris HR, Owen MJ, O'Donovan MC - Nucleic Acids Res. (2008)

Bottom Line: Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility.Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts.In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychological Medicine, Wales School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. williamsnm@cf.ac.uk

ABSTRACT
Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

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Effect of increasing both the number of reference and test assays to estimate copy number by qicPCR. hsCN was determined at 22q11 for all combinations of 1 to 5 test and reference assays in 10 patients with 22q11DS and 10 non-deleted controls. For simplicity the CV of only the 22q11DS samples is presented, however, an analogous pattern was seen in the data for the non-deleted controls.
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Figure 4: Effect of increasing both the number of reference and test assays to estimate copy number by qicPCR. hsCN was determined at 22q11 for all combinations of 1 to 5 test and reference assays in 10 patients with 22q11DS and 10 non-deleted controls. For simplicity the CV of only the 22q11DS samples is presented, however, an analogous pattern was seen in the data for the non-deleted controls.

Mentions: We next assessed the effect of increasing both the number of independent test probes and also the number of independent control probes within the same multiplex reaction. Calculating the hsDNAquant for the locus as an average of at least 4 independent test assays and then determining the mean hsCN for the locus by comparing to at least 4 reference assays resulted in a considerable improvement in data quality (Figure 4) where the low CV <0.09 and high silhouette scores (>0.77) met our predefined criteria for a genotyping assay. We performed an identical assessment of qicPCR to detect changes in copy number at 22q11 using an entirely independent multiplex panel of 22q11 test assays (qicPCR22q11b). Analysis in the same set of samples revealed that the estimates of hsCN at 22q11 by each multiplex panel of markers were similar (Figure 5) and that both CV and silhouette scores met our predefined criteria when at least 4 independent test and reference assays were used (data not presented).Figure 4.


Analysis of copy number variation using quantitative interspecies competitive PCR.

Williams NM, Williams H, Majounie E, Norton N, Glaser B, Morris HR, Owen MJ, O'Donovan MC - Nucleic Acids Res. (2008)

Effect of increasing both the number of reference and test assays to estimate copy number by qicPCR. hsCN was determined at 22q11 for all combinations of 1 to 5 test and reference assays in 10 patients with 22q11DS and 10 non-deleted controls. For simplicity the CV of only the 22q11DS samples is presented, however, an analogous pattern was seen in the data for the non-deleted controls.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2553599&req=5

Figure 4: Effect of increasing both the number of reference and test assays to estimate copy number by qicPCR. hsCN was determined at 22q11 for all combinations of 1 to 5 test and reference assays in 10 patients with 22q11DS and 10 non-deleted controls. For simplicity the CV of only the 22q11DS samples is presented, however, an analogous pattern was seen in the data for the non-deleted controls.
Mentions: We next assessed the effect of increasing both the number of independent test probes and also the number of independent control probes within the same multiplex reaction. Calculating the hsDNAquant for the locus as an average of at least 4 independent test assays and then determining the mean hsCN for the locus by comparing to at least 4 reference assays resulted in a considerable improvement in data quality (Figure 4) where the low CV <0.09 and high silhouette scores (>0.77) met our predefined criteria for a genotyping assay. We performed an identical assessment of qicPCR to detect changes in copy number at 22q11 using an entirely independent multiplex panel of 22q11 test assays (qicPCR22q11b). Analysis in the same set of samples revealed that the estimates of hsCN at 22q11 by each multiplex panel of markers were similar (Figure 5) and that both CV and silhouette scores met our predefined criteria when at least 4 independent test and reference assays were used (data not presented).Figure 4.

Bottom Line: Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility.Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts.In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychological Medicine, Wales School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, UK. williamsnm@cf.ac.uk

ABSTRACT
Over recent years small submicroscopic DNA copy-number variants (CNVs) have been highlighted as an important source of variation in the human genome, human phenotypic diversity and disease susceptibility. Consequently, there is a pressing need for the development of methods that allow the efficient, accurate and cheap measurement of genomic copy number polymorphisms in clinical cohorts. We have developed a simple competitive PCR based method to determine DNA copy number which uses the entire genome of a single chimpanzee as a competitor thus eliminating the requirement for competitive sequences to be synthesized for each assay. This results in the requirement for only a single reference sample for all assays and dramatically increases the potential for large numbers of loci to be analysed in multiplex. In this study we establish proof of concept by accurately detecting previously characterized mutations at the PARK2 locus and then demonstrating the potential of quantitative interspecies competitive PCR (qicPCR) to accurately genotype CNVs in association studies by analysing chromosome 22q11 deletions in a sample of previously characterized patients and normal controls.

Show MeSH
Related in: MedlinePlus