Extended Data Fig. 2: Error rate of the DNA duplex sequencing technology.

a) Left panel, orange bars, mutations per sequenced nucleotide detected by the DNA duplex sequencing technology in this study using the same panel of bladder genes in cord blood DNA samples from three donors. Blue bars (for comparison), mutations per sequenced nucleotide detected by a similar technology (NanoSeq; data taken from ref. ⁸²) in two cord blood samples (Supplementary Note 4). Each bar presents the mutation density computed for a separate cord blood sample, with vertical lines representing the Poisson 95% confidence intervals. The comparison of the number of observed mutations per sequenced nucleotide with those expected in cord blood DNA based on prior studies (dashed red line, Supplementary Note 4) yields an estimate of the error rate of the technology of ~4 × 10⁻⁸ per sequenced nucleotide. Right panel, comparison of the estimated error rate by both technologies with the mutations per sequence nucleotide across the 79 normal urothelial samples included in this study (rightmost red bar). It shows that the rate of errors of the DNA duplex sequencing technology used in the study is approximately 25 times smaller than the mutation density detected in the normal urothelium. (N = 3 for cord blood DNA duplex sequencing). b) Mutational profile of normal urothelium obtained through two orthogonal approaches. Top panel, profile constructed using mutations detected through laser capture microdissection (LCM) of clonal or quasi-clonal samples followed by regular shallow whole-genome sequencing (data taken from ref. ¹⁰). Bottom panel, profile constructed using mutations detected in this study from ~2 cm² brushes followed by ultradeep DNA duplex sequencing. c) Relationship between the mutation density calculated in normal bladder urothelium using two orthogonal approaches. The red dots correspond to the rate of mutations detected through laser capture microdissection of clonal or quasi-clonal samples followed by regular shallow whole-exome sequencing (WES LCMs; data taken from ref. ¹⁰). The blue dots correspond to the rate of mutations computed for the 79 samples in this study from ~2 cm² brushes followed by ultradeep DNA duplex sequencing. The trend line represented in the plot was calculated from the WES LCMs samples. For more details on the error rate of the technology, see Supplementary Note 4.