Allele-specific digital PCR enhances precision and sensitivity in the detection and quantification of copy number alterations in heterogeneous DNA samples: anin silicoandin vitrovalidation study

Abstract

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 evaluatedin silicousing a newly introduced R library ‘digitalPCRsimulations’, andin vitroby 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.

Digital PCR-based deep quantitative profiling delineates heterogeneity and evolution of uveal melanoma

“…one SNP variant) could be assured in most tumours ( Figure 4B ). This confirms our earlier observation that CNAs affecting chromosomes 3p and 8q usually involve only one of both alleles [27]. In our current cohort, we did not identify cases with isodisomy 3p, which would have been visible as a large allelic imbalance in combination with an absent or limited absolute loss of chromosome 3p.…”

“…The Gα q signalling mutations (in GNAQ , GNA11 , CYSLTR2 and PLCB4 ) and BSE mutations (in BAP1 , SF3B1 and EIF1AX ) were analysed in duplex digital PCR experiments and the measured concentrations (listed between square brackets) of the mutant and wild-type alleles were used to calculate the mutant allele fractions: These values were then used to estimate the mutant cell fractions under heterozygous conditions: In line with our recent study [27], two approaches were employed to measure CNAs. Firstly, following the classic approach, copy number values were calculated based on the measured concentrations of a DNA target and one stable diploid reference gene: The targets included genes on chromosome 3p ( PPARG ) and 8q ( PTK2 ).…”

“…In 31 tumours, chromosome 3p and/or 8q copy numbers could be determined via multiple heterozygous SNPs, which further validated the technical reproducibility of our assays and SNP-based approach ( Supplementary Figure 1C ). As this approach – in line with our recent study [27] – also led to more precise copy number measurements ( Supplementary Figure 1D ), we focussed on the SNP-based results in downstream evaluation of all copy numbers.…”

“…Extending our recent proof-of-concept study [27], chromosome 3p and 8q CNAs were analysed using two complementary digital PCR-based approaches. For the classic approach, targets on chromosome 3p and 8q were quantified against a stable reference gene using multiplex digital PCR.…”

“…Digital PCR experiments were carried out using the QX200 Droplet Digital PCR System (Bio-Rad Laboratories, Hercules, USA) following the protocols and general guidelines described and discussed earlier [21, 24–27]. In general, 20 ng DNA was analysed in a 22 uL experiment, using 11 uL ddPCR™ Supermix for Probes (No dUTP, Bio-Rad) and primers and probes in final concentrations of 900 and 250 nM for duplex experiments, or optimised concentrations for multiplex experiments.…”