A multiplex droplet digital PCR assay for chromosome copy-number determination in Candida albicans.

Chromosome copy-number variation 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 6-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 pretreatment with restriction enzymes, the assay provides accurate, reproducible chromosome-level copy-number estimates that correlate closely with whole-genome sequencing 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.