## 4 Discussion
The addition of methylation ctDNA profiling in melanoma is particularly valuable, as ctDNA has been shown to be predictive of melanoma-specific survival in high-risk stage III melanoma [13] and also predicts response and survival in advanced melanoma patients treated with molecular or immune therapies [6; 7; 48]. In these reports, however, ctDNA analysis was limited to patients with an established driver gene mutation and although highly sensitive ddPCR was used for the detection of these gene mutations, only 34% of Stage III melanoma patients had detectable ctDNA at baseline [13]. The analysis of ctDNA methylation using a panel of genes eliminates the need to define mutations using tumor tissue and increases the likelihood of detecting tumor-associated circulating DNA. The following are available online at [[http://www.mdpi.com/2076-3417/9/23/5074/s1](http://www.mdpi.com/2076-3417/9/23/5074/s1)]([http://www.mdpi.com/2076-3417/9/23/5074/s1](http://www.mdpi.com/2076-3417/9/23/5074/s1)), Figure S1: Bisulphite converted amplicon sequences, Figure S2: Comparison of the differences in the fraction of methylation determined for symmetrical CpG sites, Figure S3: Correlation of circulating free DNA methylation and ctDNA copy number in metastatic melanoma patient samples, Table S1: PCR primers, Table S2: Fraction methylation of CpG sites, Table S3: Total differentially methylated CpG sites in melanoma vs. healthy-controlsamples, Table S4: Median methylation of total differentially methylated CpG sites, Table S5: Top differentially methylated CpG sites in melanoma vs. healthy-control samples, Table S6: Median methylation of top differentially methylated CpG sites. **Author Contributions:** Conceptualization, R.J.D., J.H.L., and H.R. ; methodology, R.J.D., J.H.L., D.C., Y.W., C.P., and H.R. ; formal analysis, R.J.D., J.H.L., Y.W., C.P., and H.R. ; investigation, R.J.D., J.H.L., and D.C.; resources, R.F.K., A.M.M., M.S.C., G.V.L., and R.A.S. ; writing--original draft preparation, R.J.D., and H.R. ; writing--review and editing, R.J.D., J.H.L., D.C., Y.W., C.P., R.F.K., A.M.M., M.S.C., G.V.L., R.A.S., and H.R. ; visualization, R.J.D., J.H.L., and H.R. ; supervision, R.J.D., and H.R. ; project administration, H.R. ; funding acquisition, H.R. **Funding:** R.J.D. was supported in part by a donation to Melanoma Institute Australia from the Clearbridge Foundation. This work was also supported in part by the National Health and Medical Research Council (APP1093017 and APP1128951). H.R., R.A.S., and G.V.L. are supported by NHMRC Fellowships. G.V.L. is also supported by the Medical Foundation of The University of Sydney. A.M.M. is supported by a Cancer Institute NSW Fellowship. The Australian Genome Research Facility is supported by Bioplatforms Australia (BPA) via an Australian Government NCRIS investment (to EMBL-ABR). **Conflicts of Interest** G.V.L. receives consultant service fees from Amgen, BMS, Array, Pierre-Fabre, Novartis, Merck Sharp & Dohme (MSD), and Roche. [PERSON] is an advisory board member for BMS, MSD, Novartis, Roche, and Pierre Fabre. [PERSON] has been on advisory boards for Roche, Amgen, BMS, MSD, Novartis and TEVA and has received honoraria from MSD, BMS and Novartis. [PERSON] is an advisory board member for MSD, BMS, Novartis, Pierre-Fabre, Roche and Amgen. [PERSON] has received honoraria from MSD, Novartis, GSK, BMS, Myriad and NeraCare. BPA had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. All remaining authors have declared no conflicts of interest.