## 3 Results
### Determination of the Fraction of Circulating Free DNA Methylation  
For methylation analysis, input circulating free DNA ranged from 15-25 ng for the initial bisulfite conversion step (Table 1). Following bisulfite conversion, 7 gene target regions containing CpG islands plus spiked in lambda DNA (amplicons shown in Figure S1) were amplified and subsequently sequenced. Our workflow independently interrogated methylation of both the upper Watson and lower Crick DNA strands (Figure S1). _RASSF1_ was the only gene for which it was only feasible to design primers for the lower strand. The fraction of methylation of individual cytosines in CpG sites present in the upper and lower strand of each amplicon was determined based on sequencing reads (ratio of total methylated cytosine in a CpG motif/total reads for that cytosine; Table S2). In the case of healthy control 1, no sequencing reads were observed for the _PON3_ upper stand amplicon. Overall, the NTC sample had \(\sim\)300 reads compared to \(\sim\)50,000 reads for each DNA containing sample. At individual CpG sites, the NTC sample had 0 to 40 total reads compared to the circulating free DNA containing samples which typically had several hundred to thousands of total reads (Table S2a), and this low read depth was filtered out in our methylation analysis pipeline. The mixed methylation amplification control consisting of 1:1 mixture of unmethylated and methylated human genomic DNA showed a median CpG methylation level of 35% (Table S2b). The determination of bisulfite conversion efficiency (i.e., conversion of unmethylated cytosine residues to uracil) was based on comparing the methylated vs. unmethylated signal for the spiked-in Lambda DNA for each circulating free DNA sample (Table S2). In our data, the non-conversion ratio (Rnc %) ranged from 0.108% to 0.411%, i.e., the bisulfite conversion efficiency was highly efficient at 99.589% to 99.892%. Identification of Genes Which Discriminate for Melanoma Based on Differential Methylation of Circulating Free DNA  
A comparison of healthy controls vs melanoma patients was then undertaken based on the fraction methylation of individual CpG sites within the chosen gene targets (Figure 1). Overall, the methylation levels were variable across the 7 genes but found to be significantly elevated in melanoma patients compared to baseline levels found in healthy controls (Figure 1 and Table S3). Hierarchical clustering based on methylation levels showed that four melanoma patient samples (1, 2a, 2b and 4; with detectable ctDNA ranging from 1,615-30,350 copies/mL plasma), clustered separately from the healthy controls, whereas melanoma patient sample 3, with very low levels of ctDNA (200 copies/mL; Table 1), clustered with the healthy samples (Figure 1). Importantly, the level of methylation increased with increasing ctDNA levels, and although there were differences in the ctDNA methylation patterns in the longitudinal ctDNA samples derived from patient 2, these samples clustered closely together (Figure 1). Analysis of the DNA strand differences in methylation fractions for each target gene, excluding _RASSF1_ which only had the Watson strand amplified, illustrated that the majority of symmetrical  
Figure 1: Differentially methylated CpG sites in healthy control samples and metastatic melanoma patient samples. Heat map of the fraction of methylation determined for individual CpG sites across the indicated genes. Comparisons were based on healthy control samples versus melanoma patient samples. ctDNA levels (copy number/mL of plasma) determined for each melanoma patient samples are also shown for comparison. Details of individual CpG location sites are listed in Table S3. The scale for colors include: 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.