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.