Skip to main content

Advertisement

SpringerLink
Log in

ddpcRquant: threshold determination for single channel droplet digital PCR experiments

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Digital PCR is rapidly gaining interest in the field of molecular biology for absolute quantification of nucleic acids. However, the first generation of platforms still needs careful validation and requires a specific methodology for data analysis to distinguish negative from positive signals by defining a threshold value. The currently described methods to assess droplet digital PCR (ddPCR) are based on an underlying assumption that the fluorescent signal of droplets is normally distributed. We show that this normality assumption does not likely hold true for most ddPCR runs, resulting in an erroneous threshold. We suggest a methodology that does not make any assumptions about the distribution of the fluorescence readouts. A threshold is estimated by modelling the extreme values in the negative droplet population using extreme value theory. Furthermore, the method takes shifts in baseline fluorescence between samples into account. An R implementation of our method is available, allowing automated threshold determination for absolute ddPCR quantification using a single fluorescent reporter.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

csv:

Comma separated value

ddPCR:

Droplet digital polymerase chain reaction

dPCR:

Digital polymerase chain reaction

HTML:

HyperText Markup Language

NTC:

Negative template control

QQ plot:

Quantile–quantile plot

References

  1. Hindson CM, Chevillet JR, Briggs HA et al (2013) Absolute quantification by droplet digital PCR versus analog real-time PCR. Nat Method 10:1003–1005

    Article  CAS  Google Scholar 

  2. Sedlak RH, Jerome KR (2013) Viral diagnostics in the era of digital PCR. Diagn Microbial Infect Dis 75:1–4

    Article  CAS  Google Scholar 

  3. Pinheiro LB, Coleman VA, Hindson CM et al (2012) Evaluation of a droplet digital polymerase chain reaction format for DNA copy number quantification. Anal Chem 84:1003–11

    Article  CAS  Google Scholar 

  4. Hindson BJ, Ness KD, Masquelier DA et al (2011) High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal Chem 83:8604–10

    Article  CAS  Google Scholar 

  5. Dube S, Qin J, Ramakrishnan R (2008) Mathematical analysis of copy number variation in a DNA sample using digital PCR on a nanofluidic device. PLoS One 3, e2876

    Article  Google Scholar 

  6. Kiselinova M, Pasternak AO, De Spiegelaere W et al (2014) Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA. PLoS One 9, e85999

    Article  Google Scholar 

  7. Strain MC, Lada SM, Luong T et al (2013) Highly precise measurement of HIV DNA by droplet digital PCR. PLoS One 8, e55943

    Article  CAS  Google Scholar 

  8. Sedlak RH, Cook L, Huang M-L et al (2014) Identification of chromosomally integrated human herpesvirus 6 by droplet digital PCR. Clin Chem 60:765–72

    Article  CAS  Google Scholar 

  9. Jones M, Williams J, Gärtner K et al (2014) Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, “definetherain”. J Virol Methods 202:46–53

    Article  CAS  Google Scholar 

  10. Dreo T, Pirc M, Ramsak Z (2014) Optimising droplet digital PCR analysis approaches for detection and quantification of bacteria : a case study of fire blight and potato brown rot. Anal Bioanal Chem 406:6513–6528

    Article  CAS  Google Scholar 

  11. Buzon MJ, Massanella M, Llibre JM et al (2014) HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nat Med 16:460–65

    Article  Google Scholar 

  12. van der Sluis RM, van Montfort T, Centlivre M et al (2013) Quantitation of HIV-1 DNA with a sensitive Taqman assay that has broad subtype specificity. J Virol Methods 187:94–102

    Article  Google Scholar 

  13. Liszewski MK, Yu JJ, O’Doherty U (2009) Detecting HIV-1 integration by repetitive-sampling Alu-gag PCR. Methods 47:254–260

    Article  CAS  Google Scholar 

  14. Palmer S, Wiegand AP, Maldarelli F et al (2003) New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus Type 1 RNA in Plasma. J Clin Microbiol 41:4531–4536

    Article  CAS  Google Scholar 

  15. Fischer M, Joos B, Hirschel B et al (2004) Cellular viral rebound after cessation of potent antiretroviral therapy predicted by levels of multiply spliced HIV-1 RNA encoding nef. J Infect Dis 190:1979–1988

    Article  CAS  Google Scholar 

  16. Lizée G, Aerts JL, Gonzales MI et al (2003) Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression. Hum Gene Ther 14:497–507

    Article  Google Scholar 

  17. De Spiegelaere W, Malatinkova E, Kiselinova M et al (2013) Touchdown digital polymerase chain reaction for quantification of highly conserved sequences in the HIV-1 genome. Anal Biochem 439:201–3

    Article  Google Scholar 

  18. R Development Core Team (2014) R: A language and environment for statistical computing. R foundation for statistical computing, Vienna, Austria. http://www.R-project.org/ (Accessed January 2014)

  19. Robertson T, Cryer JD (1974) An iterative procedure for estimating the mode. J Am Stat Assoc 69(348):1012–1016

    Article  Google Scholar 

  20. Bickel DR, Fruehwirth R (2006) On a fast, robust estimator of the mode: comparisons to other robust estimators with applications. Comput Stat Data Anal 50(12):3500–3530

    Article  Google Scholar 

  21. Stephenson AG (2002) evd: Extreme Value Distributions. R News, 2(2):31–32 URL: http://CRAN.R-project.org/doc/Rnews/

  22. Malec P, Schienle M (2014) Nonparametric kernel density estimation near the boundary. Comput Stat Data Anal 72:57–76

    Article  Google Scholar 

Download references

Acknowledgments

The authors would like to acknowledge the support of following research grants: Amfar (Group funding, grant 108314-51-RGRL), HIVERA/SBO IWT (Group funding, grant 130442), FWO (Linos Vandekerckhove, grant 1.8.020.09.N.00), IWT (Pawel Bonzckowski, grant 111286; Eva Malatinkova, grant 111393), BOF (Maja Kiselinova, grant 01N02712), King Baudouin Foundation (Group funding, grant 2010-R20640-003), unrestricted grant of Bristol-Myers Squibb Belgium (Group funding), Multidisciplinary Research Partnership Bioinformatics: From Nucleotides to Networks Project (01MR0310W) of Ghent University (Group funding), and IAP research network P7/06 of the Belgian Government (Belgian Science Policy; Group funding).

Conflict of interest

The authors declare to have no competing interests.

Authors’ contributions

Performed the ddPCR runs: MK, EM, KV. Analyzed the ddPCR data: WT, PB, WDS. Developed the statistical methodology: MV, JDN, OT, WT, WDS. Wrote the R code: WT, MV. Wrote the paper: WT, WDS, MV, JDN. Critically read, reviewed, and approved the final version of the manuscript: all authors.

Author information

Authors and Affiliations

  1. Department of Internal Medicine, HIV Translational Research Unit, Ghent University and University Hospital, Depintelaan 185, De Pintepark Building, 9000, Ghent, Belgium

    Wim Trypsteen, Pawel Bonczkowski, Maja Kiselinova, Eva Malatinkova, Karen Vervisch, Linos Vandekerckhove & Ward De Spiegelaere

  2. Department of Mathematical Modelling, Statistics and Bio-informatics, Ghent University, Coupure Links 653, 9000, Ghent, Belgium

    Matthijs Vynck, Jan De Neve & Olivier Thas

  3. National Institute for Applied Statistics Research Australia (NIASRA), School of Mathematics and Applied Statistics, University of Wollongong, Northfields Avenue, Wollongong, NSW, 2522, Australia

    Olivier Thas

Authors
  1. Wim Trypsteen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Matthijs Vynck
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jan De Neve
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Pawel Bonczkowski
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Maja Kiselinova
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eva Malatinkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Karen Vervisch
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Olivier Thas
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Linos Vandekerckhove
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ward De Spiegelaere
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Linos Vandekerckhove.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 131 kb)

ESM 2

(PDF 4108 kb)

ESM 3

(PDF 313 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Trypsteen, W., Vynck, M., De Neve, J. et al. ddpcRquant: threshold determination for single channel droplet digital PCR experiments. Anal Bioanal Chem 407, 5827–5834 (2015). https://doi.org/10.1007/s00216-015-8773-4

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-015-8773-4

Keywords

Search

Navigation