A Multiplex Droplet Digital PCR Assay for Chromosome Copy Number Determination in <i>Candida albicans</i>

Chromosome copy number variation (CNV) is a major contributor to genome plasticity and adaptation in Candida albicans, a leading fungal pathogen of humans. Aneuploidy, defined as deviations from the normal diploid chromosome set, rapidly alters gene dosage and is often observed during exposure to host-imposed and antifungal stress. Accurate detection and quantification of chromosomal copy number changes are thus essential to dissect the mechanisms by which C. albicans adapts and evolves. Here, we describe the development, optimization, and validation of a six-color, 16-plex droplet digital PCR assay for simultaneous quantification of all C. albicans chromosome arms in a single reaction. Each target is detected by a unique dual-color or single-color combination of probes, enabling high-order multiplexing through binary fluorescence encoding. Following optimization of probe concentrations, PCR cycling parameters, genomic DNA extraction, and pre-treatment with restriction enzymes, the assay provides accurate, reproducible chromosome-level copy number estimates that correlate closely with WGS results across euploid and aneuploid isolates. Compared to whole-genome sequencing, the assay is rapid, cost-effective, and scalable, requiring minimal DNA input and allowing high-throughput analysis of large collections of isolates. The 16-plex assay thus provides a platform for dissecting genome instability and adaptive evolution in C. albicans.