Next generation sequencing analysis of TCR repertoire upon in vitro T-lineage differentiation of control, SCID (P1 and P2), and OS (P3)-derived iPSCs. (A) Heat map representation of percentage of TRB VJ (orientated via chromosomal 5′ to 3′ distribution) pairings among unique sequences in ISP (left) and DP (right) T-lineage cells derived from the indicated patient iPS lines. Results demonstrate 1 representative sample from 2 experiments with similar results. (B) Heat map representation of percentage of TRA VJ (orientated via chromosomal 5′ to 3′ distribution) pairings among total sequences (all δ rearrangements excluded from analysis) in ISP (left) and DP (right) T-lineage cells derived from the indicated patients’ iPS lines among unique sequences. Results demonstrate 1 representative sample from 2 experiments with similar results. (C) Quantitative PCR analysis of TRECs in control, SCID P2, and OS P3 cells. RNase P was used as an internal control for quality of genomic DNA amplification. (D) Virtual spectratyping, showing skewing in the distribution of CDR3 lengths among unique TRB or TRA sequences expressed by ISP (top) and DP (bottom) cells in SCID P1, P2, and Omenn P3 compared with control. (E) Distribution of CDR3 length for 5 more commonly expressed V genes in each sample for unique TRB sequences. (F) Distribution of CDR3 length for 5 more commonly expressed V genes in each sample for unique TRA sequences. Results demonstrate 1 representative sample from 2 experiments with similar results. RNase P, ribonuclease P.