Non-invasive determination of methylome of tumor from plasma

A method of analyzing a biological sample of an organism, the biological sample comprising cell-free DNA originating from normal cells and potentially from cells associated with cancer, the method comprising:

• analyzing a plurality of cell-free DNA molecules from the biological sample, wherein analyzing each of the plurality of cell-free DNA molecules includes:
• determining a location of the cell-free DNA molecule in a genome of the organism;
• and
• determining whether the cell-free DNA molecule is methylated at one or more sites, wherein determining whether the cell-free DNA molecule is methylated at the one or more sites comprises performing methylation-aware sequencing, and wherein performing methylation-aware sequencing comprises performing methylation-aware massively parallel sequencing, the one or more sites of each of the plurality of cell-free DNA molecules providing a plurality of sites;
• for each site of the plurality of sites, individually counting a respective number of the cell-free DNA molecules at the site that are hypermethylated;
• and
• calculating a first methylation level using the respective numbers of cell-free DNA molecules hypermethylated at the plurality of sites.