Fig. 2

A read threshold is insufficient to remove spurious barcodes. a-f Experiments with 19 clones of known barcodes, mixed in different frequencies and then diluted such that the expected total cell number per technical replicate varies from ~5 cells to ~10⁴ cells for all clones combined. Plots show number of reads in each of two technical replicates after normalization to 10⁵ reads per replicate. Green dots denote barcodes that were true, black dots denote spurious barcodes. The grey region with red border approximates a cell count of one or less, i.e., the frequency of barcode reads within this range is below what is expected for a single cell. Dashed horizontal and vertical lines and numbers alongside denote the approximate number of cells to which the normalized read numbers correspond. g-i Quantification of the performance of filtering based on a fixed read threshold (without prior normalization) when considering reference-list-based filtering as a gold standard, applied to barcode sequencing data on T cell differentiation (8) and haematopoiesis (9). Sensitivity (g), specificity (h) and precision (i) are shown as a function of the applied read threshold for four sequencing lanes (denoted by the different colors)