Abstract
Circulating cell-free tumor DNA (ctDNA) is a promising biomarker in cancer. Ultrasensitive technologies enable detection of low (< 0.1%) mutant allele frequencies, a pre-requisite to fully utilize the potential of ctDNA in cancer diagnostics. In addition, the entire liquid biopsy workflow needs to be carefully optimized to enable reliable ctDNA analysis. Here, we discuss important considerations for ctDNA detection in plasma. We show how each experimental step can easily be evaluated using simple quantitative PCR assays, including detection of cellular DNA contamination and PCR inhibition. Furthermore, ctDNA assay performance is also demonstrated to be affected by both DNA fragmentation and target sequence. Finally, we show that quantitative PCR is useful to estimate the required sequencing depth and to monitor DNA losses throughout the workflow. The use of quality control assays enables the development of robust and standardized workflows that facilitate the implementation of ctDNA analysis into clinical routine.