(a) Impact of duplex-supported barcodes on iDES noninvasive genotyping performance. Here, we performed background polishing on single-stranded barcode reads (ignoring duplex strand information) and repeated our noninvasive genotyping analysis of the 5% variant blend analyzed in Fig. 4a (right). While specificity, PPV, and NPV remained comparable to duplex-boosted iDES (bottom panel), the mean sensitivity dropped by 10% from 96% to 86% when ignoring duplexes (top panel). Thus, “duplex-boosting” improves the sensitivity of noninvasive tumor genotyping, highlighting the value of a hybrid barcoding approach within iDES. (b) Analysis of noninvasive tumor genotyping using the same variant blend in Fig. 4a,b, but added into normal control cfDNA at a 10-fold lower dilution (0.5%) and sequenced using twice the input mass (72ng DNA). Genotyping results are shown for different error suppression methods (iDES = barcoding + polishing). In all, there were 13 known alleles with externally validated AFs covered by our NSCLC selector (Supplementary Table 4). Due to the ultralow range of expected AFs (0.005% ≤ AF ≤ 0.16%), 16 additional alleles known to be present in the variant blend, but lacking external AF validation, were excluded from this analysis (Supplementary Table 4). Specificity was assessed using nearly 300 hotspot variants not present in the variant blend (Supplementary Table 4). Sensitivity and negative predictive value (NPV) were evaluated using all 13 alleles with external validation, however given the median number of hGEs recovered (=12,630), the detection-limit was determined to be 0.024% AF with 95% confidence (Statistical methods for ctDNA detection in Methods). Therefore, we also calculated detection-limit-adjusted sensitivity and NPV using only those variants with an expected AF >0.024% (n = 10 of 13; denoted with an asterisk). Genotyping was performed as described in Noninvasive tumor genotyping of hotspot alleles and selected regions in Methods. Sn, sensitivity; Sp, specificity; PPV, positive predictive value.