Fig. S3.

Criteria for validation qPCR reads. (a), Cumulative distribution of the Ct values from Tb177bp qPCR of 82 reference negative samples to stablish the Ct value cut-off. Samples were considered positive when the identity of the amplicon is been confirmed by dissociation analysis (specific melt temperature ±0.5°). The red dots represent the values of the empirical cumulative distribution function, while the black line represents its interpolation using the Gaussian smoothing proposed by GraphPad Prism 9.1.2. The Gaussian modelling of the cumulative distribution function was used to determine a threshold of Ct = 28.5 corresponding to an estimated probability of 0. The Gaussian approximation seems relevant as the overlap of the observations (in red) and the model (in black) is visually valid. (b), Dissociation analysis of the qPCR amplicons from a dilution series of TNA using cultured parasites. The specific melt temperature for the Tb177bp repeats amplicon is 78.