Sample preparation for cell-free dna analysis

A method of preparing a sample from a subject for methylation analysis, comprising:

• processing a blood sample to obtain the plasma component of the blood sample, wherein the blood sample was collected using a blood collection tube comprising an anticoagulant and an agent that inhibits the release of genomic DNA from white blood cells in the sample into the plasma component of the blood sample;
• isolating cell-free DNA (cfDNA) from the plasma component of the blood sample to provide a cfDNA sample;
• and
• digesting the cfDNA sample with one or more methylation-sensitive restriction enzymes (MSREs) and/or one or more methylation-dependent restriction enzymes (MDREs) at a temperature between about 30° C. to about 45° C. for a digestion period of between about 8hours to about 18 hours to provide a digested cfDNA sample, wherein less than 25% of the DNA molecules present in the cfDNA sample are single stranded DNA molecules during the digesting step.