Supplementary Figure 8: Theoretical detection limits of barcode-mediated error suppression methods for clinically practical quantities of cfDNA.

(a) Global error rates (x-axis) and barcode efficiencies (y-axis) for methods reported in this work (i.e., iDES, barcoding or polishing only, duplex only) compared with barcoding methods from several previous studies^12,13,14,17. Barcode efficiency reflects the number of reads required to build a barcode consensus sequence and is defined as the number of consensus sequences per read. Of note, these comparisons are not perfect since both error rate and barcode efficiency can be affected by heterogeneous sequencing amounts and sample types used in the different studies. For details on the derivation of these quantities, see Error rates and efficiencies of previous barcoding methods in Methods. In addition, the theoretical error rate of duplex sequencing is approximately equivalent to the error rate of single-stranded barcoding multiplied by itself and divided by 3 (to account for all possible base substitutions)¹². Bona fide low frequency mutations (i.e., biological background) may skew observed duplex error rates, resulting in both their overestimation and variability across studies. Data are presented as means +/- s.d. Note that due to log scaling, the limits of some error bars are shown for one direction, but not the other. (b) Comparison of all methods in a in relation to estimated detection-limit over a range of sequencing depth and cfDNA input (assuming a 90% detection probability). Sequencing was calibrated to iDES, such that the quantity of reads R needed to recover a desired number of hGEs was determined (assuming constant yield and sufficient input molecules). R was then used to calculate the number of recovered hGEs for all other methods. Modeling was performed as described in Statistical methods for ctDNA detection in Methods (and in Figs. 1a, 3d). Note that the maximum attainable detection-limit for each method is bound by its error rate. For example, barcoding-only (this study) and background polishing reach their respective maximum detection limits in this analysis. Although background polishing is not a barcoding strategy, it was included in this analysis to compare its detection-limit with other approaches. The “barcode efficiency” of polishing is identical to our barcoding approach since the same sequencing adapters were used for both.