Extended Data Fig. 6: Characterising the monoclonal lineage tracing experiment.

a. Cell doubling times measured for HEK293T and the monoclonal lineage tracing cell line (iPE2(+) LT(+)), with or without Doxycycline (Dox). The presence of Dox lengthened the cell doubling time, possibly negatively affecting the cell physiology. P values were obtained using the two-tailed Student’s t-test with Bonferroni correction: only *P < 0.05 are shown. The center and error bars are mean and standard deviations, respectively, from n = 3 independent experiments. b. Determining a set of valid TargetBCs based on frequencies. The Y-axis is on a log10-scale. Recovered TargetBCs were first ranked by their read counts to estimate multiplicity of infection (MOI) (left). Any additional TargetBCs that are 1-bp Hamming distance away from the set of 19 were corrected. We then retained 3,257 cells for which we recovered 13 of the most frequent TargetBCs (excluding one tape sequence with a corrupted type-guide) for lineage analysis (right). c. Read counts of InsertBCs observed in TAPE-1 arrays. The Y-axis is on a log10-scale. For the 3,257 selected cells, we additionally required that all observed edits were amongst the 19 most frequent InsertBCs in the overall dataset, as we presume this to be the valid set of pegRNA-defined insertional edits. d. Characterization of indel error rates of prime editing on TargetBC-5xTAPE-1 arrays. The Y-axis is on a log10-scale. Correct length insertions with prime editing are > 100-fold more likely than an insertion of a different length product. Furthermore, some of the apparent longer insertions are likely to correspond to a contraction of TAPE-1 monomer within 5xTAPE-1 before the integration, such as contraction of TGATGGTGAGCACG TAPE-1 monomer to the observed TGAGCACG 8-bp sequence appearing between two TAPE-1 monomers. e. Characterization of substitution error rates during prime editing-mediated insertion of the GGA key sequence on TargetBC-5xTAPE-1 arrays. The X-axis is on a log10-scale. Correct insertions are > 100-fold more likely than insertions with substitution errors. The most frequent class of errors are transition errors, and these may be occurring during PCR amplification or sequencing-by-synthesis of cDNA amplicons, rather than during prime editing. Data in panel (b) to (e) is generated from n = 1 monoclonal lineage experiment, followed by n = 1 single-cell RNA-seq data collection. f. A lineage tree constructed by order-aware UPGMA for a clade of 81 cells drawn from the larger tree. Numbers next to branching points denote bootstrap values out of 100 resamplings. The 59 sites of 13 TargetBC-associated tape arrays are represented to the right, with InsertBCs colored by edit identity. Cells are identified by the 16-bp CellBCs (10X Chromium v3 chemistry) listed on the far right.