Accuracy in Copy Number Calling by qPCR and PRT: A Matter of DNA

Fernández-Jiménez, Nora; Castellanos–Rubio, Ainara; Plaza-Izurieta, Leticia; Gutiérrez, Galder; Irastorza, Iñaki; Castaño, Luís; Vitoria, Juan Carlos; Bilbao, José Ramón

doi:10.1371/journal.pone.0028910

Cited by 29 publications

(28 citation statements)

References 20 publications

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“…Also, efficient predigestion of gDNA improves ddPCR assay accuracy by facilitating template access. 32 Indeed, assay variability was reduced and greater consistency in estimated VCN values was observed with up to a minimum of approximately 1 ng of template (Fig. 5b).…”

Section: Discussionmentioning

confidence: 86%

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

Lin

Okumura

Yatsuda³

et al. 2016

Human Gene Therapy Methods

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Stable gene transfer into target cell populations via integrating viral vectors is widely used in stem cell gene therapy (SCGT). Accurate vector copy number (VCN) estimation has become increasingly important. However, existing methods of estimation such as real-time quantitative PCR are more restricted in practicality, especially during clinical trials, given the limited availability of sample materials from patients. This study demonstrates the application of an emerging technology called droplet digital PCR (ddPCR) in estimating VCN states in the context of SCGT. Induced pluripotent stem cells (iPSCs) derived from a patient with X-linked chronic granulomatous disease were used as clonable target cells for transduction with alpharetroviral vectors harboring codon-optimized CYBB cDNA. Precise primer–probe design followed by multiplex analysis conferred assay specificity. Accurate estimation of per-cell VCN values was possible without reliance on a reference standard curve. Sensitivity was high and the dynamic range of detection was wide. Assay reliability was validated by observation of consistent, reproducible, and distinct VCN clustering patterns for clones of transduced iPSCs with varying numbers of transgene copies. Taken together, use of ddPCR appears to offer a practical and robust approach to VCN estimation with a wide range of clinical and research applications.

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Section: Discussionmentioning

confidence: 86%

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

Lin

Okumura

Yatsuda³

et al. 2016

Human Gene Therapy Methods

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“…24 However, Fernandez-Jimenez et al demonstrated that both techniques can produce comparable DEFB4 results with the use of optimum DNA normalisation and high-quality genomic DNA. 28 Nevertheless, it is known that DEFB4 copy numbers vary between ethnicities; 29 this might account for the differences observed between the studied Iraqi and Korean populations. 24 Genetic heterogeneity was observed in the β-defensin gene cluster among 67 populations; this may have been due to selection.…”

Section: Discussionmentioning

confidence: 94%

Association of Higher Defensin β-4 Genomic Copy Numbers with Behçet’s Disease in Iraqi Patients

Hameed¹,

Jaradat²,

Al-Musawi³

et al. 2015

SQUMJ

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abstract:Objectives: Behçet's disease (BD) is an immune-mediated small vessel systemic vasculitis. Human β-defensins are antimicrobial peptides associated with many inflammatory diseases and are encoded by the β-defensin family of multiple-copy genes. However, their role in BD necessitates further investigation. The aim of the present study was to investigate the possible association of BD in its various clinical forms with defensin β-4 (DEFB4) genomic copy numbers. Methods: This case-control study was conducted from January to September 2011 and included 50 control subjects and 27 unrelated Iraqi BD patients registered at Baghdad Teaching Hospital, Bagdad, Iraq. Copy numbers of the DEFB4 gene were determined using the comparative cycle threshold method by duplex real-time polymerase chain reaction technology at the Department of Dermatology of Jena University Hospital, Jena, Germany. Results: DEFB4 genomic copy numbers were significantly higher in the BD group compared to the control group (P = 0.010). However, no statistically significant association was found between copy numbers and clinical variables within the BD group. Conclusion: The DEFB4 copy number polymorphism may be associated with BD; however, it is not associated with different clinical manifestations of the disease.

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“…Additionally, it has been suggested that real-time QPCR will perform comparably to PRT if the concentration of DNA samples is normalized to a uniform concentration, and the efficiency between the test and reference loci differ by less than 5% [18]. With knowledge of these potential variables on assay performance, we felt that it would be unfair to perform a method comparison on DNA samples that were subject to such limitations.…”

Section: Discussionmentioning

confidence: 99%

Technical considerations for genotyping multi-allelic copy number variation (CNV), in regions of segmental duplication

et al. 2014

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BackgroundIntrachromosomal segmental duplications provide the substrate for non-allelic homologous recombination, facilitating extensive copy number variation in the human genome. Many multi-copy gene families are embedded within genomic regions with high levels of sequence identity (>95%) and therefore pose considerable analytical challenges. In some cases, the complexity involved in analyzing such regions is largely underestimated. Rapid, cost effective analysis of multi-copy gene regions have typically implemented quantitative approaches, however quantitative data are not an absolute means of certainty. Therefore any technique prone to degrees of measurement error can produce ambiguous results that may lead to spurious associations with complex disease.ResultsIn this study we have focused on testing the accuracy and reproducibility of quantitative analysis techniques. With reference to the C-C Chemokine Ligand-3-like-1 (CCL3L1) gene, we performed analysis using real-time Quantitative PCR (QPCR), Multiplex Ligation-dependent Probe Amplification (MLPA) and Paralogue Ratio Test (PRT). After controlling for potential outside variables on assay performance, including DNA concentration, quality, preparation and storage conditions, we find that real-time QPCR produces data that does not cluster tightly around copy number integer values, with variation substantially greater than that of the MLPA or PRT systems. We find that the method of rounding real-time QPCR measurements can potentially lead to mis-scoring of copy number genotypes and suggest caution should be exercised in interpreting QPCR data.ConclusionsWe conclude that real-time QPCR is inherently prone to measurement error, even under conditions that would seem favorable for association studies. Our results indicate that potential variability in the physicochemical properties of the DNA samples cannot solely explain the poor performance exhibited by the real-time QPCR systems. We recommend that more robust approaches such as PRT or MLPA should be used to genotype multi-allelic copy number variation in disease association studies and suggest several approaches which can be implemented to ensure the quality of the copy number typing using quantitative methods.

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Accuracy in Copy Number Calling by qPCR and PRT: A Matter of DNA

Cited by 29 publications

References 20 publications

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

Application of Droplet Digital PCR for Estimating Vector Copy Number States in Stem Cell Gene Therapy

Association of Higher Defensin β-4 Genomic Copy Numbers with Behçet’s Disease in Iraqi Patients

Technical considerations for genotyping multi-allelic copy number variation (CNV), in regions of segmental duplication

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