Fig. 1 Cell-free DNA a) entering the bloodstream b) laboratory workflow
Blood plasma contains short fragments of cell-free DNA, thought to be released through apoptotic cell cleavage. Cell-free DNA is typically highly fragmented, in the range of 100–200 bp. Additionally, in cancer patients, circulating DNA from lysed tumour cells (ctDNA) is present (Fig. 1a). The concentration of ctDNA can be as little as 0.1% of all cell-free DNA in patients with early-stage disease. Targeting specific oncogenes in cell-free DNA from patients allows mutations to be detected and tracked in order to help understand disease progression. To help detect low-frequency variants, UMIs can be added to DNA fragments prior to sequence capture using biotinylated baits. After PCR and sequencing, the UMIs can be used to cluster and polish the reads resulting in a high-accuracy single molecule consensus sequence (Fig. 1b).