Benchmarking digital PCR partition classification methods with empirical and simulated duplex data.

Digital PCR (dPCR) is a highly accurate technique for the quantification of target nucleic acid(s). It has shown great potential in clinical applications, like tumor liquid biopsy and validation of biomarkers. Accurate classification of partitions based on end-point fluorescence intensities is crucial to avoid biased estimators of the concentration of the target molecules. We have evaluated many clustering methods, from general-purpose methods to specific methods for dPCR and flowcytometry, on both simulated and real-life data. Clustering method performance was evaluated by simulating various scenarios. Based on our extensive comparison of clustering methods, we describe the limits of these methods, and formulate guidelines for choosing an appropriate method. In addition, we have developed a novel method for simulating realistic dPCR data. The method is based on a mixture distribution of a Poisson point process and a skew-$t$ distribution, which enables the generation of irregularities of cluster shapes and randomness of partitions between clusters ('rain') as commonly observed in dPCR data. Users can fine-tune the model parameters and generate labeled datasets, using their own data as a template. Besides, the database of experimental dPCR data augmented with the labeled simulated data can serve as training and testing data for new clustering methods. The simulation method is available as an R Shiny app.

Measuring DNA quality by digital PCR using probability calculations.

BACKGROUND: Accurate methods to assess DNA integrity are needed for many biomolecular methods. A multiplex digital PCR (dPCR) method designed for interspaced target sequences can be used to assess sequence integrity of large DNA strands. The ratio of single positive partitions versus double positive partitions is then used to calculate the sheared DNA strands. However, this simple calculation is only valid with low DNA concentration. We here describe a method based on probability calculations which enables DNA quality analysis in a large dynamic range of DNA concentrations. RESULTS: Known DNA integrity percentages were mimicked using artificial double stranded DNA in low, intermediate and high DNA concentration scenarios, respectively 600, 12500 and 30000 copies of DNA per reaction. At low concentrations both methods were similar. However, at the intermediate concentration (12500 copies per reaction) the ratio based method started producing a larger error than the proposed probability calculation method with a mean relative error of 20.7 and 16.7 for the Bruner and the proposed method respectively. At the high concentration (30000 copies per reaction) only the proposed method provided accurate measurements with a mean relative error of 60.9 and 9.3 for the ratio based and the proposed method respectively. Furthermore, while both methods have a bias, it is constant for the proposed method, while it decreases with the integrity of the DNA for the ratio based method. The probability calculation equation was extended to 4 dimensions and a proof of concept experiment was performed, the data suggested that the 4 dimensional equation is valid. SIGNIFICANCE AND NOVELTY: We here validate a method of estimating DNA integrity with dPCR using multiple probe combinations, allowing fast and flexible DNA integrity analysis. Additionally, we extend the method from 2 to 4 plex for more accurate DNA integrity measurements.

Digital PCR Partition Classification.

BACKGROUND: Partition classification is a critical step in the digital PCR data analysis pipeline. A range of partition classification methods have been developed, many motivated by specific experimental setups. An overview of these partition classification methods is lacking and their comparative properties are often unclear, likely impacting the proper application of these methods. CONTENT: This review provides a summary of all available digital PCR partition classification approaches and the challenges they aim to overcome, serving as a guide for the digital PCR practitioner wishing to apply them. We additionally discuss strengths and weaknesses of these methods, which can further guide practitioners in vigilant application of these existing methods. This review provides method developers with ideas for improving methods or designing new ones. The latter is further stimulated by our identification and discussion of application gaps in the literature, for which there are currently no or few methods available. SUMMARY: This review provides an overview of digital PCR partition classification methods, their properties, and potential applications. Ideas for further advances are presented and may bolster method development.

Different local, innate and adaptive immune responses are induced by two commercial Mycoplasma hyopneumoniae bacterins and an adjuvant alone.

Introduction: Enzootic pneumonia still causes major economic losses to the intensive pig production. Vaccination against its primary pathogen, Mycoplasma hyopneumoniae, is carried out worldwide to control the disease and minimize clinical signs and performance losses. Nonetheless, the effects of both infection with, and vaccination against Mycoplasma hyopneumoniae on the innate and adaptive immune responses remain largely unknown. Therefore, we conducted a study in which piglets were injected once with a commercial bacterin V1 or V2, or the adjuvant of V1 (A) to investigate their effect on local, innate and adaptive immune responses. Methods: Three weeks after vaccination, piglets were challenge infected with M. hyopneumoniae and euthanized four weeks later to assess vaccine efficacy via macroscopic and microscopic evaluation of lung lesions. Blood and broncho-alveolar lavage fluid (BAL) samples were collected to measure antibody responses, cellular immunity, BAL cytokine levels and BAL M. hyopneumoniae DNA load as well as cytokine secretion by monocytes. Results: After vaccination, proliferation of antigen-specific CD3+ T cells and a higher percentage of TNF-α+ CD8+, and TNF-α+ and TNF-α+IFN-γ+ CD4+CD8+ T cells was seen in V1, while proliferation of or a significant increase in cytokine production by different T cell subsets could not be observed for animals from V2. Interestingly, LPS-stimulated blood monocytes from V1 and A secreted less IL-10 on D7. After challenge, higher levels of IgA, more IL-10 and less IL-1ß was detected in BAL from V1, which was not observed in V2. Animals from A had significantly more IL-17A in BAL. The macroscopic lung lesion score and the M. hyopneumoniae DNA load at euthanasia was lower in V1, but the microscopic lung lesion score was lower in both vaccinated groups. Discussion: In conclusion, these results indicate that the two commercial bacterins induced different local and adaptive immune responses, that the adjuvant alone can reduce anti-inflammatory innate immune responses, and that both vaccines had a different efficacy to reduce Mycoplasma-like lung lesions and M. hyopneumoniae DNA load in the lung.

Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA.

The development of an HIV-1 cure is hampered by the existence of a persistent (latent) reservoir that contains a small proportion of replication-competent intact proviruses which refuels viral replication upon treatment discontinuation. Therefore, an accurate evaluation and quantification of these (intact) proviruses is essential to determine the efficacy of HIV-1 cure strategies which aim to eliminate this reservoir. Here, we present two triplex digital PCR assays which resulted from a combination of two existing methods, the IPDA (a 2-colour digital PCR based method) and Q4PCR assays (4 colour qPCR method), and tested the functionality on a three-colour digital PCR platform. In the present paper, we provide a step-by-step experimental protocol for these triplex digital PCR assays and validate their performance on a latently infected Jurkat cell-line model and HIV-1 patient samples. Our data demonstrates the potential and flexibility of increasing the number of subgenomic regions of HIV-1 within the IPDA to acquire sensitive detection of the HIV-1 reservoir while benefitting from the advantages of a dPCR setup.