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Liquid biopsies are beginning to screen for DNA methylation in cancer-derived DNA and one-use case is that epigenetic modifications are being used to assign a tissue of origin to the cancer to direct confirmatory diagnostic procedures. However, accurate assessment of methylation is not trivial, and methods like bisulfite sequencing can result in DNA damage in the sample. Furthermore, obtaining sufficient amounts of cell-free circulating tumor DNA (ctDNA) for assay development, validation, and performance monitoring is a challenge and reference materials for establishing the analytical validity of measurements of CpG methylation are not widely available. Here, we describe new ctDNA-sized reference materials designed to address the challenges associated with assessing DNA methylation of ctDNA. 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