Next, we compared the transcriptomes of the four probands to those of the unaffected family members (two to three iPSC clones per person) at two time points, TD11 and TD31. Differential gene expression (DGE) between the probands and the respective fathers, used as sex-matched normal controls, identified 1062 differentially expressed genes (DEGs) at TD11 and 2203 DEGs at TD31 (see Table S2A), hinting at a possibly divergent developmental trajectory between controls and probands. Validation by qPCR of a subset of the DEGs identified by RNAseq revealed a 0.98 correlation coefficient between log2 fold changes from the two techniques and 100% concordance in direction of change (see Table S2B). The individual-to-individual (biological) variability, as modeled in RNA-seq DGE analyses, is clearly narrow (Table S2). The iPSC line-to-line variability is also quite low, as shown by the boxplots of correlation coefficients within each individual (intra) and across individuals (inter) (Figure S4). In fact, the variability between lines from the same individual is lower than the variability between lines across individuals (Figure S4A–C). This reproducibility is likely due to the robustness of our telencephalic organoid preparation, which selects for forebrain progenitors while allowing spontaneous 3-D organization.
