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Quality control of digital PCR assays and platforms

Matthijs Vynck (UGent) , Jo Vandesompele (UGent) and Olivier Thas (UGent)
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Abstract
Digital polymerase chain reaction (digital PCR, dPCR) is a direct nucleic acid quantification method, thus requiring no standard curves unlike quantitative real-time PCR (qPCR). Nevertheless, evaluation of the linear dynamic range, accuracy, and precision of an assay or platform is recommended, as there are several potential causes of important non-linearity, bias, and imprecision. Ignoring these quality issues may lead to erroneous quantification. This necessitates an approach akin to the construction of standard curves. We study the pitfalls associated with the evaluation of such an experiment, and provide guidelines for the assessment of linearity, accuracy, and precision in dPCR experiments. We present simulation results and a case study supporting the importance of a thorough evaluation. Further, typically presented plots and statistics may not reveal problems with linearity, accuracy, or precision. We find that a robust weighted least-squares approach is highly advisable, yet may also suffer from an inflated false-positive rate. The proposed assessments are also applicable to other analyses, such as the comparison of results obtained from qPCR and dPCR. A web tool for quality evaluation, dPCalibRate, is available.
Keywords
Digital PCR, Quality control, Linearity, Accuracy, Precision, REAL-TIME PCR, GUIDELINES MINIMUM INFORMATION, QUANTIFICATION, HETEROSKEDASTICITY, PUBLICATION, SAMPLES, DNA

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MLA
Vynck, Matthijs, et al. “Quality Control of Digital PCR Assays and Platforms.” ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 409, no. 25, 2017, pp. 5919–31, doi:10.1007/s00216-017-0538-9.
APA
Vynck, M., Vandesompele, J., & Thas, O. (2017). Quality control of digital PCR assays and platforms. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 409(25), 5919–5931. https://doi.org/10.1007/s00216-017-0538-9
Chicago author-date
Vynck, Matthijs, Jo Vandesompele, and Olivier Thas. 2017. “Quality Control of Digital PCR Assays and Platforms.” ANALYTICAL AND BIOANALYTICAL CHEMISTRY 409 (25): 5919–31. https://doi.org/10.1007/s00216-017-0538-9.
Chicago author-date (all authors)
Vynck, Matthijs, Jo Vandesompele, and Olivier Thas. 2017. “Quality Control of Digital PCR Assays and Platforms.” ANALYTICAL AND BIOANALYTICAL CHEMISTRY 409 (25): 5919–5931. doi:10.1007/s00216-017-0538-9.
Vancouver
1.
Vynck M, Vandesompele J, Thas O. Quality control of digital PCR assays and platforms. ANALYTICAL AND BIOANALYTICAL CHEMISTRY. 2017;409(25):5919–31.
IEEE
[1]
M. Vynck, J. Vandesompele, and O. Thas, “Quality control of digital PCR assays and platforms,” ANALYTICAL AND BIOANALYTICAL CHEMISTRY, vol. 409, no. 25, pp. 5919–5931, 2017.
@article{8528592,
  abstract     = {{Digital polymerase chain reaction (digital PCR, dPCR) is a direct nucleic acid quantification method, thus requiring no standard curves unlike quantitative real-time PCR (qPCR). Nevertheless, evaluation of the linear dynamic range, accuracy, and precision of an assay or platform is recommended, as there are several potential causes of important non-linearity, bias, and imprecision. Ignoring these quality issues may lead to erroneous quantification. This necessitates an approach akin to the construction of standard curves. We study the pitfalls associated with the evaluation of such an experiment, and provide guidelines for the assessment of linearity, accuracy, and precision in dPCR experiments. We present simulation results and a case study supporting the importance of a thorough evaluation. Further, typically presented plots and statistics may not reveal problems with linearity, accuracy, or precision. We find that a robust weighted least-squares approach is highly advisable, yet may also suffer from an inflated false-positive rate. The proposed assessments are also applicable to other analyses, such as the comparison of results obtained from qPCR and dPCR. A web tool for quality evaluation, dPCalibRate, is available.}},
  author       = {{Vynck, Matthijs and Vandesompele, Jo and Thas, Olivier}},
  issn         = {{1618-2642}},
  journal      = {{ANALYTICAL AND BIOANALYTICAL CHEMISTRY}},
  keywords     = {{Digital PCR,Quality control,Linearity,Accuracy,Precision,REAL-TIME PCR,GUIDELINES MINIMUM INFORMATION,QUANTIFICATION,HETEROSKEDASTICITY,PUBLICATION,SAMPLES,DNA}},
  language     = {{eng}},
  number       = {{25}},
  pages        = {{5919--5931}},
  title        = {{Quality control of digital PCR assays and platforms}},
  url          = {{http://doi.org/10.1007/s00216-017-0538-9}},
  volume       = {{409}},
  year         = {{2017}},
}

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