## 3 Results
### Comparison of Circulating Free DNA Methylation and Copy Number Levels in Melanoma Patients  
We noted that the level of circulating free DNA methylation increased with increasing ctDNA (Figure 1), and we further examined the correlation between ctDNA levels (Table 1) and circulating free DNA methylation levels for each gene in the melanoma patient samples. Significant correlation was found for _PON3_ and _MEOX2_ suggesting that measuring the methylation of these gene targets using circulating free DNA can be an accurate surrogate of ctDNA copy number (Figure 3). The other five gene targets had lower correlation coefficients which were not significant (Figure S3).  
Figure 2: Circulating free DNA methylation can discriminate between healthy and melanoma patients. ROC curves for each gene target were based on median methylation using the fraction of methylation for each CpG site in both the upper and lower stand amplicons.  
Evaluation of Methylated CpG Sites in Circulating Free DNA as a Biomarker for Detecting for Metastatic Melanoma  
We next sought to compare 64 individual CpG sites in the _GJB2_, _HOXA9_, _MEOX2_, _OLIG3_, _PON3_ and _RASSF1_ genes. These 64 CpGs were most significantly differentially methylated when melanoma patient samples were compared to the healthy control samples (FDR adjusted \(p\)-value \(<\) 0.1; Tables S3 and S5). Hierarchical clustering using these 64 CpG sites confirmed that melanoma patient sample 3, with very low levels of ctDNA, remain clustered with the healthy controls (Figure 4A) as seen in our initial analyses (Figure 1). Of the remaining melanoma patient samples, all with detectable ctDNA, melanoma patient sample 2a-EDT (with the lowest level of detectable ctDNA) was more distantly related to the remaining patient cluster including melanoma patient samples 1, 2a-PRE and 4 (Figure 4A). Our initial analysis showed patient samples 2a-PRE and 2b-EDT clustering together and patient samples 1 and 4 clustering together (Figure 1).  
We then performed ROC analysis based on the median circulating free DNA methylation values calculated from the fraction methylation of the 64 most differentially methylated individual CpG sites (shown in Figure 4A) for each healthy and melanoma sample (Table S6). This again demonstrated significant hypermethylation in patients with melanoma compared with healthy control (AUC = 0.92; Figure 4B).  
Figure 3: Correlation of circulating free DNA methylation and ctDNA copy number in metastatic melanoma patient samples. Methylation was based on median methylation using the fraction of methylation for each CpG site in both the upper and lower stand amplicons for each gene target. ctDNA copy number/mL of plasma was determined using ddPCR. The dashed lines represent the average of the median methylation levels for healthy control samples.  
Figure 4: Most significant differentially methylated CpG sites in healthy control samples and metastatic melanoma patient samples. (**A**) Heat map of the fraction of methylation determined for individual CpG sites across the indicated genes in the order highest to lowest t-test value. Comparisons were based on healthy control samples versus melanoma patient samples. ctDNA levels (copy number/mL of plasma) determined for melanoma patient samples are also shown for comparison. The scale for colors includes: blue for row minimum levels of methylation of CpG sites in indicated genes or ctDNA  
levels (copy number/mL plasma) and red for row maximum levels of methylation of CpG sites in indicated genes or ctDNA levels (copy number/mL plasma). A black box indicates data was not determined. The chromosome position and number is indicated for each CpG site. (**B**) ROC curve was based on median methylation using the fraction of methylation for each of the most significant individual differentially methylated CpG sites for healthy control samples and melanoma patient samples.