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Michał Burdukiewicz 1,*,Jim Hugget2, Alexandra Whale2, Boris Fehse3, Piotr Sobczyk4, Paweł Mackiewicz1, Andrej-Nikolai Spiess5, Peter Schierack 6 , Stefan Rödiger 6 1 Department of Genomics, University of Wrocław, Poland 2 Molecular and Cell Biology Team, LGC, Teddington, United Kingdom 3 Research Department Cell and Gene Therapy, Department of SCT, University Medical Center Hamburg-Eppendorf, Hamburg, Germany 4 Faculty of Pure and Applied Mathematics, Wrocław University of Science and Technology, Wrocław, Poland 5 University Medical Center Hamburg-Eppendorf, Hamburg, Germany 6 Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, Senftenberg, Germany |
Abstract
Digital PCR (dPCR) is a variant of PCR, where the PCR ampli- fication is conducted in multiple small volume reactions (termed partitions) instead of a bulk. The dichotomous status of each par- tition (positive or negative amplification) is used for absolute quantification of the template molecules by Poisson transforma- tion of the proportion of positive partitions. The vast expansion of dPCR technology and its applications has been followed by the development of statistical data analysis methods. Yet, the software landscape is scattered, consisting of scripts in various programming languages, web servers with narrow scopes or closed source vendor software packages, that are usually tightly tied to their platform. This leads to unfavourable environments, as results from differ- ent platforms, or even from different laboratories using the same platform, cannot be easily compared with one another.
To address these problems, we developed the dpcReport web server that provides an open-source tool for the analysis of dPCR data. dpcReport provides a streamlined analysis framework to the dPCR community that is compatible with the data output (e.g., CSV, XLSX) from different dPCR platforms (e.g., Bio-Rad QX100/200, Biomark). This goes beyond the basic dPCR data analysis with vendor-supplied softwares, which is often limited to the compu- tation of the mean template copy number per partition and its uncertainty. dpcReport gives users more control over their data analysis and they benefit from standardization and reproducible analysis.
Our web server analyses data regardless of the platform ven- dor or type (droplet or chamber dPCR). It is not limited to the commercially available platforms and can also be used with exper- imental systems by importing data through the universal REDF format, which follows the IETF® RFC 4180 standard. dpcReport provides users with advanced tools for data quality control and it incorporates statistical tests for comparing multiple reactions in an experiment, currently absent in many dPCR-related soft- ware tools. dpcReport provides users with advanced tools for data quality control. The conducted analyses are fully integrated within extensive and customizable interactive HTML reports including figures, tables and calculations. To improve reproducibility and transparency, a report may include snippets in the programming language R enabling an exact reproduction of the analysis per- formed by dpcReport through functions from the dpcR package. Our software follows the standardized dPCR nomenclature of the dMIQE guidelines. Since the vast functionality offered by dpcRe- port may be overwhelming at first, our web server is extensively documented.
The server is freely accessible at: http://www.smorfland.uni. wroc.pl/shiny/dpcReport/.
http://dx.doi.org/10.1016/j.bdq.2017.02.077
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