title: ddpcRquant: threshold determination for single channel droplet digital PCR experiments.
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.
authors.affiliation: Department of Internal Medicine, HIV Translational Research Unit, Ghent University and University Hospital, Depintelaan 185, De Pintepark Building, 9000, Ghent, Belgium.
authors.forename: Wim
authors.initials: W
authors.lastname: Trypsteen
authors.forename: Matthijs
authors.initials: M
authors.lastname: Vynck
authors.forename: Jan
authors.initials: J
authors.lastname: De Neve
authors.forename: Pawel
authors.initials: P
authors.lastname: Bonczkowski
authors.forename: Maja
authors.initials: M
authors.lastname: Kiselinova
authors.forename: Eva
authors.initials: E
authors.lastname: Malatinkova
authors.forename: Karen
authors.initials: K
authors.lastname: Vervisch
authors.forename: Olivier
authors.initials: O
authors.lastname: Thas
authors.forename: Linos
authors.initials: L
authors.lastname: Vandekerckhove
authors.forename: Ward
authors.initials: W
authors.lastname: De Spiegelaere
mesh_terms: D005453:Fluorescence; D016133:Polymerase Chain Reaction / Q000379:methods*