Supplementary Figure 11: Specificity of biopsy-free genotyping and correlation of hotspot mutation frequency with NSCLC disease stage.

(a) Analysis of the specificity of noninvasive tumor genotyping using normal control cfDNA samples. To compare specificity among error-suppression methods, we only analyzed normal controls that were not used for building the background-polishing database (n = 18; Methods, Supplementary Table 4). (b) Comparison of error suppression methods for the mean number of variants detected per cfDNA sample in 18 normal controls (same as in a) and all 24 pretreatment NSCLC samples with matching tumor biopsies (Supplementary Table 2). Group comparisons were performed using a two-sided Wilcoxon rank sum test (NS, not significant). Data are expressed as means +/- 95% confidence intervals. (c) Percentage of pretreatment NSCLC cfDNA samples (n = 24), organized by tumor stage, with at least one variant noninvasively detected by iDES-enhanced CAPP-Seq (related to Fig. 4d). (d) AFs for all variants noninvasively detected in cfDNA samples from 24 NSCLC patients (with sequenced tumors) using iDES-enhanced CAPP-Seq (Supplementary Table 4). Samples are ranked from left to right by decreasing mean AF. Error bars denote AF range. Tick marks (x-axis) indicate individual cfDNA samples. A list of 292 candidate hotspot variants was used for the analyses in a-d, excluding those specific to the variant blend analyzed in Fig. 4a,b (Supplementary Table 4). Genotyping was performed as described in Noninvasive tumor genotyping of hotspot alleles and selected regions in Methods.