Genome integrity of induced pluripotent stem cells (iPSCs) has been extensively studied in recent years, but it is still unclear whether iPSCs contain more genomic variations than cultured somatic cells. One important question is the origin of genomic variations detected in iPSCs-whether iPSC reprogramming induces such variations. Here, we undertook a unique approach by deriving fibroblast subclones and clonal iPSC lines from the same fibroblast population and applied next-generation sequencing to compare genomic variations in these lines. Targeted deep sequencing of parental fibroblasts revealed that most variants detected in clonal iPSCs and fibroblast subclones were rare variants inherited from the parental fibroblasts. Only a small number of variants remained undetectable in the parental fibroblasts, which were thus likely to be de novo. Importantly, the clonal iPSCs and fibroblast subclones contained comparable numbers of de novo variants. Collectively, our data suggest that iPSC reprogramming is not mutagenic.iPSCs | fibroblasts | reprogramming | genomic variation | exome sequencing G enomic integrity of human induced pluripotent stem cells (iPSCs) is an unresolved question and a crucial issue for iPSCs-based therapeutics. To understand the scope of acquired genetic changes that occur during iPSC generation, previous studies have compared single nucleotide variants (SNVs), copy number variations (CNVs), and chromosomal rearrangements in iPSCs to donor somatic cells or embryonic stem cells (ESCs) using various assays, including SNP array and next-generation sequencing methods (1-6). Whole genome or exome sequencing (WGS/ WES) of parental and iPSC pairs have demonstrated that there are, on average, 6-12 coding SNVs per iPSC line (1, 7-9) with varying theories regarding when these SNVs arose in the iPSCs. Most reports attributed de novo SNVs or CNVs in iPSCs to reprogramming-induced stress or long-term in vitro culture (2,3,7,8,(10)(11)(12)(13)(14). However, several studies also suggested that many of the "de novo" variants were either inherited rare preexisting variations in the founder source cells or benign variants regardless of reprogramming method used (1,9,15,16).To determine whether iPSCs are inherently more likely to accumulate mutations and further elucidate the origin of genomic variants present in iPSCs, we have undertaken a unique approach by establishing clonal fibroblast subclones and iPSCs from the same fibroblast population to directly assess whether the reprogramming process leads to more mutations by next-generation sequencing. De novo mutations, which were not detected in the starting pooled parental fibroblasts, were identified in each daughter cell line, both fibroblast subclones and iPSCs. These putative de novo mutation sites were then deep sequenced in the parental fibroblast population to determine whether they were present at low frequency as mosaic variants. In addition, highresolution single nucleotide polymorphism (SNP) arrays were used to search for de novo CNVs in both fibrobla...
