Allele-specific digital PCR enhances precision and sensitivity in the detection and quantification of copy number alterations in heterogeneous DNA samples: an in silico and in vitro validation study

The analysis of genetic variation is of crucial importance in cancer care. Measuring copy number alterations, however, remains challenging in heterogenous DNA samples, such as (liquid) biopsies. Using digital PCR, these alterations are classically studied by comparing the abundances of the target of interest to a stable genomic reference. Alternatively, copy numbers may be quantified based on the allelic (im)balance of a heterozygous common single-nucleotide polymorphism (SNP). In this study, the accuracy, practicability, precision and sensitivity of both approaches are evaluated in silico using a newly introduced R library ‘digitalPCRsimulations’, and in vitro by analysing control samples and uveal melanoma specimens in duplex and multiplex experimental setups. Our analyses show that both methodologies are equally effective, as long as a stable reference is identified (classic approach) and the allelic imbalance is caused by the loss or gain/amplification of only one of both alleles (SNP-based approach). Though, heterogeneous copy number alterations can be measured with more precision and sensitivity using the SNP-based approach. In DNA from formalin-fixed and paraffin-embedded samples, the latter approach can also overcome technical artefacts causing inconsistencies between DNA amplicons. In conclusion, the limits of detecting copy number alterations in heterogeneous DNA can be improved using the SNP-based approach. Consequently, an increasing number of clinical samples may be successfully analysed, providing novel potential for the identification, prognostication and management of cancer. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement Rogier J. Nell was financially supported by the European Union's Horizon 2020 Research and Innovation Program under grant agreement number 667787 (UM Cure 2020 project). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Biobank Committee and Medisch Ethische Toetsingscommissie (= ethics committee) of Leiden University Medical Center gave ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript.