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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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(A) Effect of increasing the number of reference samples to estimate copy number by qicPCR. (B) Effect of increasing the number of test assays to estimate copy number by qicPCR. Blue vertical bars represent the range of hsCN determined by qicPCR for 22q11DS patients and controls while histograms represent the CV. The ranges of silhouette scores determined at each parameter are displayed as black vertical bars. For each measure the mean is indicated as a small horizontal bar.
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Figure 3: (A) Effect of increasing the number of reference samples to estimate copy number by qicPCR. (B) Effect of increasing the number of test assays to estimate copy number by qicPCR. Blue vertical bars represent the range of hsCN determined by qicPCR for 22q11DS patients and controls while histograms represent the CV. The ranges of silhouette scores determined at each parameter are displayed as black vertical bars. For each measure the mean is indicated as a small horizontal bar.

Mentions: We reasoned that the measurement error in the hsCN determined by qicPCR could be reduced by; (1) averaging multiple independent unlinked reference loci and/or (2) averaging multiple independent test probes targeting the same test locus. We therefore set out to systematically assess the variability associated with these parameters. Estimating hsCN using a single test probe with reference to an increasing number of independent control assays (1 to 5) resulted in tighter confidence intervals for the average estimated hsCN for both 22q11DS and non-deleted samples (Figure 3A). However, the modest reduction in the average CV (22q11DS 0.25–0.19; controls 0.28–0.21) and a minor increase in the silhouette scores [0.16 (−0.03min to 0.32max) to 0.32 (0.26min–0.36max)] (Figure 3A), did not achieve the required performance.Figure 3.


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)

(A) Effect of increasing the number of reference samples to estimate copy number by qicPCR. (B) Effect of increasing the number of test assays to estimate copy number by qicPCR. Blue vertical bars represent the range of hsCN determined by qicPCR for 22q11DS patients and controls while histograms represent the CV. The ranges of silhouette scores determined at each parameter are displayed as black vertical bars. For each measure the mean is indicated as a small horizontal bar.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: (A) Effect of increasing the number of reference samples to estimate copy number by qicPCR. (B) Effect of increasing the number of test assays to estimate copy number by qicPCR. Blue vertical bars represent the range of hsCN determined by qicPCR for 22q11DS patients and controls while histograms represent the CV. The ranges of silhouette scores determined at each parameter are displayed as black vertical bars. For each measure the mean is indicated as a small horizontal bar.
Mentions: We reasoned that the measurement error in the hsCN determined by qicPCR could be reduced by; (1) averaging multiple independent unlinked reference loci and/or (2) averaging multiple independent test probes targeting the same test locus. We therefore set out to systematically assess the variability associated with these parameters. Estimating hsCN using a single test probe with reference to an increasing number of independent control assays (1 to 5) resulted in tighter confidence intervals for the average estimated hsCN for both 22q11DS and non-deleted samples (Figure 3A). However, the modest reduction in the average CV (22q11DS 0.25–0.19; controls 0.28–0.21) and a minor increase in the silhouette scores [0.16 (−0.03min to 0.32max) to 0.32 (0.26min–0.36max)] (Figure 3A), did not achieve the required performance.Figure 3.

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