Box 1 Recommendations for DNA methylation assay design

Genomic location

 • Before designing a DNA methylation biomarker assay, make a rational choice for the genomic location of the assay

    • For example, sequencing or publicly available data such as TCGA to identify the optimal genomic location

Primer- and probe design

 • Ensure the primers and probes are able to discriminate unmethylated from methylated DNA

    • Appropriate amount of CpG dinucleotides and non-CpG cytosines in primers and probe

 • Ensure the primers and probes have the ability to anneal efficiently

    • CpG dinucleotides at most 3’ end of primer, primer length, avoiding premature quenching of probe fluorophore

 • Ensure primers and probes are designed as an assay, rather than single primers and probes

    • Similar Tm between primers and appropriate Tm of probe relative to the Tm of the primers

 • Consider sample type in assay development

    • For liquid biopsies, the total assay amplicon size should be maximum 120 bp

Assay optimization

 • In silico analysis of assay

 • Optimize PCR conditions

    • Use appropriate controls

 • Perform pilot studies

    • Determine cutoff