Supplementary Figure 12: Empirical spike analysis to determine the detection-limit of iDES-enhanced CAPP-Seq and duplex sequencing.

Analysis of the detection-limit of each method in this study. Acoustically shorn DNA from a hyper-mutated glioblastoma (GBM) tumor was added into healthy donor-derived cfDNA in four 32ng dilutions, ranging from 2.5 in 10⁴ molecules down to 2.5 in 10⁶ molecules. All mixtures were assessed in two technical replicates except for the lowest fraction, which was assessed in four replicates. A custom selector targeting all 1,502 non-silent mutations identified in this tumor was used. (a) Comparison of error-suppression methods (with/without barcoding and with/without background polishing) applied to the spike series. Data with two technical replicates are presented as means with minimum-to-maximum ranges. Data with four technical replicates (lowest spike only) are presented as medians +/- interquartile range. (b) Analysis of one technical replicate from the spike series in a using 20 mutations randomly selected from the pool of 1,502 total mutations. Random sampling was repeated 50 times and the results are presented as means +/- 95% confidence intervals. (c) Same as a, but for duplex molecules only. The detection-limit (dashed line) was determined by pooling duplex sequence data from 12 normal control cfDNA samples and by calculating the mean AF of the 1,502 GBM mutations. The presentation of data and error bars is identical to panel a. (d) Observed versus expected mutation counts for each sample plotted in c. The number of expected mutations was estimated based on the expected fraction of tumor-derived DNA molecules in each sample and the number of duplex hGEs observed (Statistical methods for ctDNA detection in Methods). Additional details related to these analyses are provided in ctDNA detection limits for iDES, duplex sequencing, and other methods in Methods.