Sequencing analysis of ccfDNA
SOMATIC MUTATIONS
Raw sequencing reads were processed and aligned using the Subread alignerTo calculate the ctDNA content, SNVs/indels with a variant allele fraction (VAF) of ≥0.1% were averaged to generate the mean VAF values of each patient. ctDNA concentrations were expressed in haploid genome equivalents (hGE) per mL of plasma (hGE/mL) and calculated by multiplying the mean ctDNA VAF (determined by CheCUP panel sequencing) or the predicted TFx (determined by sWGS) by the concentration of cell-free DNA (cfDNA) (pg/mL of plasma), as determined by Qubit fluorometry. Based on the assumption that most somatic mutations are heterozygous the resulting values were then divided by 3.3 (for mean ctDNA VAF) or 6.6 (for TFx), as each haploid genomic equivalent weighs 3.3 pg, as previously described by Scherer et al., with the expected relationship between mean ctDNA VAF and TFx being ‘mean VAF*2=TFx’. If both mean ctDNA VAF and TFx could be determined in a sample, mean ctDNA VAF had priority for the calculation of ctDNA concentrations due to the higher sensitivity of the targeted NGS approach. For follow-up samples from one patient, ctDNA contents were calculated by consistently using only one of the sequencing approaches.