## 1 Introduction  
Molecular analyses of circulating DNA, RNA, proteins, vesicles or cells as liquid biopsies is a rapidly developing modality for monitoring disease progression, treatment response and tumor heterogeneity [1,2]. Circulating tumor DNA (ctDNA) is highly fragmented DNA that is derived from tumor cells and tends to increase in abundance with increasing total patient tumor burden [3,4].  
Baseline and on-treatment ctDNA is predictive of response to targeted and immunotherapies in many cancers [5; 6; 7; 8], can monitor tumor heterogeneity and the expansion of therapy resistant tumor subclones [9; 10; 11], and predicts recurrence and survival following curative resection [12; 13].  
The detection and monitoring of ctDNA using mutation-detection techniques such as droplet digital PCR (ddPCR) and targeted next generation sequencing panels has been well documented [14; 15]. One remaining challenge remains is the sensitivity of ctDNA detection, as it often accounts for less than 0.1% of the total circulating free DNA, has a short half-life of only a few hours and amounts can vary depending on cancer type and stage, tumor burden and location, vascularity and cellular turnover [3]. As a result, ctDNA does not reliably detect patients with early stage cancer [3] or with post-treatment minimal residual disease [16] and often requires a known target mutation for detection, e.g., BRAF V600E.  
Analysis of epigenetic DNA modifications, such as methylation, is a promising approach for improving ctDNA sensitivity given the high degree of methylation concordance between circulating free DNA and tissue-derived genomic DNA [17; 18; 19; 20; 21]. Moreover, epigenetic changes are important in tumor development and progression as these epigenetic changes regulate gene expression, often via the methylation of cytosine residues within CpG islands of gene promoter regions [22]. Analysis of DNA methylation requires bisulfite conversion which enables subsequent discrimination of methylated and unmethylated cytosine residues. Although bisulfite conversion can result in significant loss of DNA due to fragmentation and degradation, a recent study confirmed that the EZ DNA Methylation-Direct kit was optimal for bisulfite conversion and recovery of low molecular weight DNA such as circulating free DNA [23].  
In this study, we developed an efficient ctDNA methylation analysis workflow that utilizes the EZ DNA Methylation-Direct kit for conversion of small amounts of low molecular weight DNA (\(\sim\)20 ng) followed by MiSeq next generation sequencing to examine the methylation status of individual CpG sites within a panel of seven genes. Methylation of these genes (_GlyB2_, _HOXA9_, _MEOX2_, _OLIG3_, _PON3_, _RASSF1_ and _TEAP2B_) has been associated with melanoma progression and/or poor survival [24; 25]. In contrast to previous studies analyzing circulating free DNA methylation [26; 27; 28; 20], we examined the methylation of symmetrical CpG sites in both DNA strands to accurately quantify the level of gene methylation. Given the clinical potential of ctDNA detection in melanoma [2; 29] and the fact that promoter methylation contributes to the progression of melanoma [30], we focused on monitoring the methylation status of circulating free DNA in metastatic melanoma patients to identify melanoma-specific methylation patterns.