Analytical Performance of the Avida Duo Assay for Simultaneous Mutation and Methylation Profiling in Circulating Cell-Free DNA

Background: Circulating cell-free DNA (cfDNA) enables minimally invasive tumour genomic profiling, yet simultaneous interrogation of mutations and DNA methylation remains limited by assay complexity and input constraints. Methods: Here, we evaluate the Agilent Avida Duo system, a single workflow integrating high-sensitivity mutation detection with targeted DNA methylation analysis. We analysed 21 stage III and IV melanoma patient samples. Results: The Avida Duo mutation assay detected mutant allele frequencies (MAFs) down to 0.05% and identified tumour-associated mutations in all melanoma patients, including within GC-rich regions such as the TERT promoter. Optimisation of the Avida Duo mutation workflow, using TapeStation-quantified cfDNA and reduced amplification cycles, improved library consistency without compromising sensitivity. Methylation profiling of the melanoma baseline cohort with the Avida Duo methylation panel showed high concordance with QIAseq targeted methylation results, with the mean cfDNA fraction methylation ranging from 0.051–0.079 in most patients and reaching 0.249 in the patient with the highest ctDNA burden (MAFs up to 35.4%). Conclusions: Our findings demonstrate that the Avida Duo workflow enables simultaneous, high-resolution detection of mutation and methylation profiles from a single cfDNA sample, streamlining processing and enhancing molecular insight for clinical and translational applications.