Schematic diagram of sequencing determined SNP detection at TI sites. Forward and reverse sequencing in duplicate was performed on the ∼2-kb PCR products amplified from genomic DNA of donor C TI gene-corrected p47-CGD iPSCs. PCR products were generated from 2 primer sets, one amplifying from 5′ of the first distinguishing SNP in intron 1 to the puromycin cassette and the other from the puromycin cassette to 3′ of the last distinguishing SNP in intron 2 of NCF1 or its pseudogenes. The highlighted regions are color-coded based on the SNP’s gene identity with blue as NCF1 (1), orange as NCF1B pseudogene (1B), and yellow as NCF1C pseudogene (1C). Some SNPs represent only NCF1, NCF1B, or NCF1C, while others can represent 2 different potential combinations. The single-color rows for 16 of 18 clones indicate unambiguous concordance across all SNPs in both primer products, allowing single-locus assignment of the TI. Crosshatch rows for 2 clones represent discordance of SNPs across or between the primer products, suggestive of the presence of TI at 2 loci. The first PCR product contained 3 potential SNPs, and the second PCR product contained exon 2 and multiple SNPs.