Figure 5

Mutational characteristics of somatic variants identified in iPSC cultures by exome sequencing. Stacked bar chart for the 14 primary RiPSC and SiPSC cultures from 4 individuals with passage numbers between 7 and 15 showing the relative number of variants partitioned (A) using SnpEff software annotated by variant impact group (HIGH = green, MODERATE = blue, LOW = light green), (B) by variant type (SNV = light green, MNP = blue, indel = green) and (C) by mutational subtype (transitions in brownish, transversions in greyish turquoise) of the SNVs in each iPSC sample. For A and B absolute variant counts are in the bars. (D) Distribution of three different SNV classifier scores represented as violin plots with median and quartiles. Red line represents the respective cutoff values (CADD = 20, M-CAP = 0.025, REVEL = 0.5). (E) Dot-plot showing the distribution of allele fraction (AF) in the analyzed iPSC cell cultures (x-axis) and their corresponding fibroblast culture (y-axis) with each point representing a variant shaded by read coverage in the iPSC exome (bright = low, dark = high read coverage at the respective variant position). Dotted vertical lines mark the expected AF for a heterozygous fixed variant (0.5) and typical variabilities seen in short read sequencing (0.3 to 0.7). (F) Dot-plot showing the relation between read coverage in the analyzed iPSC cultures and AF in the corresponding fibroblast culture. Dots are grouped and colored by fibroblast AF (no evidence in fibroblast = grey, ≤5.0% = blue, ≤10.0% = orange, >10% = green). The blue line represents the linear regression model fit (formula y ~ log(x); R² = 0.202, p-value < 2.2e–16). The black line represents the theoretical AF in the fibroblast culture which is detectable at the respective coverage with a probability of 0.426 (variants with no evidence in fibroblast = 546, variants with at least 1 read in fibroblast = 405; 405/(405 + 546) ≈ 0.426) under a simple binomial draw model where one read is considered as sufficient evidence in the fibroblast. The red dotted line marks read coverage of below 20 where a high sampling variance is expected.