iPSCs and fibroblast subclones from the same
fibroblast population contain comparable levels
of sequence variations
Erika M. Kwona, John P. Connellya,1, Nancy F. Hansenb, Frank X. Donovanc, Thomas Winklerd, Brian W. Davise,
Halah Alkadia, Settara C. Chandrasekharappac, Cynthia E. Dunbard, James C. Mullikinb, and Paul Liua,2
a
Oncogenesis and Development Section, National Human Genome Research Institute, NIH, Bethesda, MD 20892; bComparative Genomics Unit, NIH
Intramural Sequencing Center, National Human Genome Research Institute, NIH, Bethesda, MD 20892; cGenomics Core, National Human Genome Research
Institute, NIH, Bethesda, MD 20892; dHematology Branch, National Heart Lung and Blood Institute, NIH, Bethesda, MD 20892; and eCancer Genetics and
Comparative Genomics Branch, National Human Genome Research Institute, NIH, Bethesda, MD 20892
Edited by Rudolf Jaenisch, Whitehead Institute, Cambridge, MA, and approved January 6, 2017 (received for review September 26, 2016)
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 iPSCswhether 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 sequenc-
ing methods (16). Whole genome or exome sequencing (WGS/
WES) of parental and iPSC pairs have demonstrated that there
are, on average, 612 coding SNVs per iPSC line (1, 79) 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, 1014). 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 ac-
cumulate 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 reprog-
ramming process leads to more mutations by next-generation se-
quencing. 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, high-
resolution single nucleotide polymorphism (SNP) arrays were used
www.pnas.org/cgi/doi/10.1073/pnas.1616035114
to search for de novo CNVs in both fibroblast and iPSC clones,
and all detected CNVs were subjected to validation using sec-
ondary methods. Our data show that iPSCs do not contain more
genomic variations than the fibroblast subclones, suggesting that
the iPSC reprogramming is not mutagenic. In addition, more than
90% of the putative new mutations in the daughter lines (both
iPSCs and fibroblast subclones) preexist in the parental fibroblast
population at very low frequencies.
GENETICS
Results
Establishment of Single-Cell Clonal Fibroblast and iPSC Lines. Clonal
fibroblast lines and iPSC lines were derived from two independent
skin fibroblast cultures (Fig. 1A). We will from here on refer to
both fibroblast subclones and iPSC lines as daughter lines. Pa-
rental fibroblast line 1 (PF1) was obtained from a patient with
familial platelet disorder (FPD), who carried a Y260X mutation in
the RUNX1 gene (17), and the second parental fibroblast line
(PF2) was obtained from a clinically healthy donor (CTRL-1) (18).
Early passage fibroblasts (passage 4) were single-cell sorted into
96-well plates and clonally expanded to establish the subclones and
to obtain sufficient amounts of genomic DNA (gDNA) for whole
exome sequencing and SNP genotyping analyses (Fig. 1B). In
Significance
One important unsolved question in the stem cell field is, do
induced pluripotent stem cells (iPSCs) have more mutations than
other cultured somatic cells because of the reprogramming
process? In this work, we took a novel approach to interrogate
the genome integrity of iPSCs by comparing mutational load of
clonal fibroblast lines and iPSC lines derived from the same fi-
broblast parental cells. Whole exome sequencing demonstrates
that iPSCs and clonal fibroblasts have comparable numbers of
new mutations, as compared with their parental fibroblasts.
Deep, targeted resequencing also shows that greater than 90%
of these mutations are random, preexisting sequence variants in
small subsets of the parental fibroblast population. Our data
strongly suggest that reprogramming process is not mutagenic.
Author contributions: E.M.K., J.P.C., S.C.C., and P.L. designed research; E.M.K., J.P.C., F.X.D.,
and H.A. performed research; T.W. contributed new reagents/analytic tools; E.M.K., N.F.H.,
F.X.D., B.W.D., S.C.C., C.E.D., J.C.M., and P.L. analyzed data; and E.M.K., C.E.D., and P.L. wrote
the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The sequences reported in this paper have been deposited in the dbGaP
database, www.ncbi.nlm.nih.gov/gap (accession no. phs001277.v1.p1).
1
Present address: Genome Engineering and iPSC Center, Washington University, St. Louis,
MO 63110.
2
To whom correspondence should be addressed. Email: pliu@mail.nih.gov.
This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.
1073/pnas.1616035114/-/DCSupplemental.
PNAS Early Edition | 1 of 6
 APooled B
Pre-existing variant
Fibroblasts gDNA from PF1 and PF2 sample cohorts
New variant
(N=2)

WES SNP array

Single fibroblast OSKM
cell isolation Reprogramming
Identify variants with 0 Find unique CNVs
reads in parental cells in sublines

Targeted deep FISH, ddPCR of CNV

resequencing regions in parental
..... ..... Expansion cells and sublines

Identify pre-existing
and de novo variants
Clonally expanded fibroblast
subclones (N=15) Clonal iPSC lines (N=5)

Fig. 1. Overall experimental design. (A) Schematic of establishment of clonal fibroblast subclones and clonal iPSCs from pooled parental fibroblast pop-
ulations. (B) Flowchart of experimental design to detect de novo mutations in daughter lines by sequencing and SNP array methods.

total, we obtained 10 fibroblast subclones from PF1 and 5 fi-
broblast subclones from PF2. Generation of clonal iPSC lines
from each starting skin fibroblast population was previously
reported (17, 18). We obtained two clonal iPSC lines from PF1
and three clonal iPSC lines from PF2, analyzing both early and
late passage iPSCs to compare accumulation of somatic muta-
tions during long-term in vitro culture. Schematic representation
of each daughter line and its relationship to the parental fibro-
blasts are depicted in Fig. 1.
parental fibroblasts (SI Appendix, Tables S2 and S3). Among the
450 variants from the PF1 cohort, 315 (70.0%) were present in
only one daughter line, whereas 135 (30%) were shared by at least
two daughter lines, including 4 variants that were shared by all
daughter lines (Fig. 2C and SI Appendix, Table S4). Similarly, the
PF2 samples had 224 variants (60.5%) that were unique to one
daughter line and 146 variants (39.5%) shared by at least two

Whole Exome Sequencing Discovered Putative New Variant Daughter

A
162
180
Lines. WES was conducted to detect genomic variants in iPSCs or Shared Variants
160
fibroblast subclones that were not present in the parental fibro- Unique Variants
140
118

blast populations (Fig. 1B). We obtained high-quality sequence 61

Variants (N)

120
reads with an average exome coverage rate of 85% for PF1 and 100

83
50

94% for PF2 sample sets (SI Appendix, Table S1). The average
76

68

68

67
66

80
60

59
coverage depth was 88-fold and 63-fold for PF1 and PF2, re- 56
101

60

53
39
spectively (PF1 range: 56 to 105; PF2 range: 54 to 71). To
58

53
46

53

54
40
49

48
68

50

identify somatic variations in individual daughter lines, we looked

12 7 32
20

30
20
18

for variants that were completely absent in respective parental

3 15

6 15

7 13

12 11
10 11

2 6

0
fibroblasts using Shimmer (19). We identified a wide range of
14

26

3
1E

1F

1G

1G

2A

2D

2F
2Y

2Y

1G

2B

2F
somatic variants in each daughter line, ranging from 39 to 162
19

19

putative new variants per line (Figs. 2A and 3A). iPSCs Fibroblast Subclones
Variant frequency (VF) spectrums of PF1 and PF2 fibroblast
subclones and iPSCs are depicted in Figs. 2B and 3B, respectively. B VF =0.05 C Total variants = 450
100

Interestingly, most of the variants had VFs around 0.5 in the

Unique
daughter lines, leading us to hypothesize that these variants were
80

rare preexisting mutations in parental fibroblasts or resulted from 315

Shared
increased postmutation fitness. On the other hand, there were low
Variants (N)
60

135
frequent variants (VF < 0.05, vertical lines in Figs. 2B and 3B) in
daughter lines (8% of the variants detected in PF1 daughter lines
40

and 2% of the variants for PF2 daughter lines), which may rep- D Fibroblast
resent variants that were either de novo somatic mutations that subclones
20

iPSCs
occurred spontaneously during in vitro culture or preexisting var-
iants with reduced fitness (Figs. 2B and 3B and SI Appendix, Tables 88 81 281
0

S2 and S3). 0.0 0.2 0.4 0.8 1.0

We compared mutation load between fibroblast subclones and
clonal iPSCs and observed that iPSCs had a 1.3- to 1.7-fold in-
creased number of putative new variants compared with fibroblast
subclones (97 vs. 73 for PF1 and 111 vs. 66 for PF2; Figs. 2A and
3A). Differences in the mutation load of iPSCs and fibroblast sub-
clones for both PF1 and PF2 are shown to be marginally significant
with a P value of 0.048 (two-tailed t test).
Shared Genomic Variants Among iPSCs and Fibroblast Subclones
Derived from Skin Fibroblasts. A total of 450 (425 SNVs and 25
indels) and 370 (357 SNVs and 13 indels) variants, respectively,
were detected in the PF1 and PF2 daughter lines but not in the
0.6
Frequency
Fig. 2. Somatic mutations detected by whole exome sequencing in parental
fibroblast 1 (PF1) clonal iPSCs and fibroblast subclones. (A) Total numbers of
putative de novo variants in individual daughter lines are shown in the bar
graph. Orange bar indicates variants that are shared among PF1 daughter lines
and unique variants to individual daughter lines are shaded in blue. Total
number of variants for each daughter line is indicated on Top of each bar.
(B) Variant frequency (VF) spectrum of putative de novo variants. If more than
one daughter line shared the variant, maximum VF is used. Gray vertical line
indicates VF = 0.05. (C) Venn diagram of putative de novo variants that are
either unique (blue) or shared (yellow). (D) Venn diagram of number of de
novo variants that are common between iPSCs and fibroblast subclones.

2 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1616035114 Kwon et al.

 Over 90% of the Putative New Variants Are Preexisting, Rare Variants
A

119
111
120 101 Shared Variants in Parental Fibroblasts. To determine whether any of the putative
Unique Variants
new variants were present in the parental fibroblasts at low fre-
100
quency, we performed targeted deep resequencing using Nim-

55

76
blegen custom capture kit at these variant sites. Approximately

72
80
67
Variants (N)

67

66
90% of targeted variant sites, which included both the unique and
72

60 shared variants, were resequenced, with average sequence depth

48
49

38
of 4,300 and 10,000 for PF1 and PF2, respectively (SI Appendix,

69

20
40

66
Tables S7 and S8). The untargeted sites have either failed custom
64
44

20 capture design due to repeats in the target regions or insufficient

29

28

28
23
read depth (<100).

7
0
1
iPSC2 iPSC3 iPSC26 1B04 1G04 1H06 3A08 3C03 Our deep targeted resequencing revealed that 9095% of the
putative de novo variants, including almost all shared variants,
iPSCs Fibroblast Subclones could be detected in parental cells at low frequency, with median
VFs of 0.00758 and 0.00063 in PF1 and PF2, respectively (Figs. 4A
B VF =0.05 C Total variants =370 and 5A). As expected, we observed much higher median VFs for
70

putative de novo variants that were shared by multiple daughter

Unique lines compared with variants that were unique to a single daughter
60

224 Shared line (Fig. 4 B and C for PF1 and Fig. 5 B and C for PF2). The
50
Variants (N)

146 finding that the shared variants exist in parental cells at higher
frequency is consistent with the fact that these variants have an
40

D increased chance of being inherited by multiple daughter lines.

30

Fibroblast Moreover, it is still possible that these variants provide functional

iPSCs
20

subclones advantage, such as increased reproductive fitness in clonal growth.

GENETICS
However, genes that contain the shared variants did not show
10

enrichment for any biological processes.

0

0.0 0.2 0.4 0.6 0.8 1.0
Frequency
Fig. 3. Somatic mutations detected by whole exome sequencing in parental
fibroblast 2 (PF2) clonal iPSCs and fibroblast subclones. (A) Total numbers of
putative de novo variants in individual daughter lines are shown in the bar
graph. Orange bar indicates variants that are shared among PF2 daughter lines
and unique variants to individual daughter lines are shaded in blue. Total
number of variants for each daughter line is indicated on Top of each bar.
Variants from early and late passage iPSC lines are combined into one bar
graph. (B) Variant frequency (VF) spectrum of putative de novo variants. If
more than one daughter line shared the variant, maximum VF is used. Gray
vertical line indicates VF = 0.05. (C) Venn diagram of de novo variants that are
either unique (blue) or shared (yellow). (D) Venn diagram of number of de
novo variants that are common between iPSCs and fibroblast subclones.
daughter lines, including 5 variants shared by all daughter lines
(Fig. 3C and SI Appendix, Table S5). Interestingly, 18% of
variants (81/450 for PF1 and 64/370 for PF2, respectively) were
shared between iPSCs and fibroblast subclones in both sample
sets (Figs. 2D and 3D). These shared variants, especially ones
shared among all daughter lines, were most likely rare variants
present in a subset of cells of the parental fibroblast population
and did not show enrichment of any biological process (SI Ap-
pendix, Table S6). In parental cells, the variants were undetect-
able by WES, possibly due to low variant frequency; however,
because each daughter line was clonally expanded, the variant
frequency rises above the detection level in these daughter lines.
Consistent with this hypothesis, the shared variants in daughter
lines were identified as true heterozygotes with median VFs of
0.35 for PF1 and 0.48 for PF2.
We checked for differences in sequencing read depth between
shared and unique variant sites to eliminate the possibility of lower
coverage at the shared sites of the parental populations. We found
that the median sequencing coverage was slightly higher at shared
variant sites compared with the unique variant sites (PF1: 111 vs.
63; PF2: 57 vs. 49). Both unique and shared variants had roughly
50% exonic variants and VFs were similar among shared variants
regardless of their genomic location (SI Appendix, Fig. S1 AD for
PF1 and Fig. S2 AD for PF2).
iPSCs and Fibroblast Subclones Have Similar Numbers of de Novo
Variants. Targeted deep sequencing revealed that only 45 and 18
putative de novo variants remained undetectable in PF1 and PF2
parental fibroblasts, respectively (SI Appendix, Tables S7 and S8).
Importantly, the numbers of these likely de novo mutations were
no longer different between the iPSCs and the fibroblast subclones
(average of 3.7 per iPSC line vs. 4 per fibroblast subclone for PF1
and 2.4 per iPSC line vs. 2 per fibroblast subclone for PF2).
Moreover, we observed no difference in median VFs between
preexisting and de novo variants in daughter lines from both PF1
and PF2 (SI Appendix, Fig. S3 A and B).
Interestingly, we found that the transition (Ts) mutation type was
more common for preexisting variants, whereas transversion (Tv)
was more common for de novo variants (average Ts/Tv ratio of 1.5
for preexisting variants and 0.46 for de novo variants). For the PF1
cohort, most preexisting variants had either G > A or C > T
changes, which are characteristic types of UV-induced mutations,
whereas most de novo variants had either C > A or G > T changes
(Fig. 4 D and E). The number of de novo variants for the PF2
cohort was too small to make this comparison (Fig. 5 D and E).
These data suggest that the cells may have undergone different
mechanisms to acquire somatic changes than the ones that are
inherited from parental cells.
Majority of de Novo Variants Are Random Somatic Changes Present in
Daughter Lines. We further characterized the de novo variants
identified by targeted resequencing to investigate whether any
variants had increased fitness that may lead to clonal selection of
cells favoring survival or reprogramming into pluripotency. First,
evaluating variants that are located in the coding region demon-
strated that iPSCs lines contained fewer mutations in the coding
region (4 coding variants/5 iPSC lines = 0.8 new mutations per
clone) than the fibroblast subclones (22 coding variants/15 sublones =
1.47 new mutations per clone), indicating no selective increase of
mutations in the iPSCs (SI Appendix, Table S9). Secondly, all de
novo variants were unique to one daughter line with the exception
of one variant in the COL1A1 gene, which was shared by PF2
fibroblast subclones 1H06 and 1G04. We suspect that this shared
variant is a preexisting variant in PF2 parental fibroblasts and
undetected during resequencing due to low targeted capture cov-
erage at this site (375, compared with 10,000 average coverage

Kwon et al. PNAS Early Edition | 3 of 6

A deep resequencing (VF < 0.0001). We observed that more than
half of the de novo variants have VFs > 0.4 in the daughter lines
(SI Appendix, Fig. S3 A and B), which suggest that these variants
originate during the very early passages of the daughter lines.

Copy Number Variation Analysis in the iPSCs and Fibroblast Subclones.

Putative new CNVs in the daughter lines were investigated using
high-density SNP arrays (Illumina HumanOmniExpressExome-
8v1.1_B). The SNP call rates were greater than 99% and all sam-
ples met desired quality control criteria except for one fibroblast

A
250 Pre existing in parental cells
B C 200
VF median = 0.00063

Variants (N)
150

100

50

0
0.00 0.05 0.10 0.15 0.20 0.25 0.30
Frequency

D B C Shared
Unique
100
150
VF median = 0.00045 80 VF median = 0.0012

Variants (N)
Variants (N)

100 60

40
50
20

0 0
0.010 0.030 0.00 0.10 0.20
E Frequency Frequency

D Pre existing mutation type

T

70
A
G

60
Variants (N)

50
C>A
T>C

G>T
40

T>G
Fig. 4. Variant frequencies and mutation types of preexisting and de novo
G>C
C>G

30
T

variants determined by targeted deep sequencing of PF1 samples. (A) Overall

C

VF spectrum of preexisting variants in the parental fibroblasts. VF spectrum of 20

T>A

unique variants (B) and shared variants (C) in the parental fibroblasts. Distri- 10
bution of variants for each mutation type for preexisting variants (D) and de 0
novo variants (E). Pink bars indicate transition mutations, and gray bars in- # of SNVs 48 56 66 29 12 16 35 6 19 18 28 24
dicate transversion mutations.
E mutation type
G>A

5
Variants (N)

at other sites). Thirdly, we performed gene enrichment analysis 4

G>C
G

G>T

T>G

using Gene Ontology (geneontology.org/) and DAVID (https:// 3

C>A
C
A>G

david.ncifcrf.gov/home.jsp) of de novo or inherited variants for 2

C
C>T

both PF1 and PF2 and observed no enrichment of any biological 1

A

0
pathways among the de novo variants. It should be noted that we # of SNVs 1 4 0 0 1 0 1 1 2 2 2 2
did not observe any enrichment of any biological pathways among
the preexisting variants either. Fig. 5. Variant frequencies and mutation types of preexisting and de novo
Collectively, these results suggest that these de novo variants variants determined by targeted deep sequencing of PF2 samples. (A) Overall
VF spectrum of preexisting variants in the parental fibroblasts. VF spectrum of
present in the fibroblast sublones and clonal iPSCs were acquired
unique variants (B) and shared variants (C) in the parental fibroblasts. Distri-
randomly during in vitro culture. Of course it cannot be ruled out bution of variants for each mutation type for preexisting variants (D) and de
completely that these variants are still preexisting variants in the novo variants (E). Pink bars indicate transition mutations, and gray bars in-
parental fibroblast cells at levels undetectable even with targeted dicate transversion mutations.

4 of 6 | www.pnas.org/cgi/doi/10.1073/pnas.1616035114 Kwon et al.

subclone, 2B12, which was excluded from further analysis. Among
the group, 55% of the daughter lines from PF1 (1/2 iPSCs and 5/9
fibroblast subclones) and 25% of the daughter lines from PF2 (1/3
iPSCs and 1/5 fibroblast subclones) had no new CNV, and iPSCs
and fibroblast subclones had comparable numbers of putative new
CNVs (one to three CNV events per clone) (SI Appendix, Table
S10). Putative somatic copy number changes included sizes ranging
from 18 kb to 800 kb, and numbers of CNV gain or loss events were
also comparable in iPSCs and fibroblast subclones. We observed
one homozygous deletion event in a fibroblast subclone (2A6)
containing a single gene, AUTS2. The rest of them were all het-
erozygous, and three deletion and six duplication events were ob-
served among the PF1 cohort and six deletions and three
duplications were observed among the PF2 cohort. Interestingly,
two CNVs (deletion of chr1: 46,287,86946,673,924 and duplica-
tion of chr2: 206,452,025206,725,525) were shared between two
fibroblast subclones, 1H06 and 1G04, suggesting that these are rare
CNVs preexisting in parental somatic cells that were inherited by
both subclones. Moreover, other larger putative structural varia-
tions were detected, including a mosaic chromosomal event in the
fibroblast subclone 2A6 (20% mosaicism of t(5;11)(q35;q13), SI
Appendix, Fig. S5) and a mosaic chromosome duplication at the
highest passage (n = 39) of an iPSC line in our study (chr12:1
133,810,935, SI Appendix, Fig. S6).
Evaluation of Putative Structural Variations as Preexisting Variations.
We set out to test for the presence of putative CNVs in the pa-
rental fibroblasts and to validate identified CNVs in daughter lines
by fluorescent in situ hybridization (FISH) for larger structural
variations. Using BAC clones as FISH probes, we validated
structural variations including the mosaic t(5;11)(q35;q13) in PF1-
2A6 at the comparable frequency of 20 (SI Appendix, Fig. S5).
However, our FISH results did not reveal the presence of any of
these structural changes in the parental fibroblasts, which could be
due to low sensitivity of the FISH method. Therefore, we further
investigated the preexistence of these changes in the parental fi-
broblasts using digital droplet PCR (ddPCR), which allows abso-
lute quantification of CNVs at 0.1% sensitivity of detection. We
were able to successfully target six of the putative de novo CNV
regions for PF1 daughter lines and for two putative de novo CNVs
shared by PF2 fibroblast subclones 1H06 and 1G04, because these
shared CNVs are likely to be preexisting variations (SI Appendix,
Figs. S7 and S8). Among the targeted putative de novo CNVs, one
amplification event on chr4: 86,288,42386,420,622 was detected
in the parental fibroblast cells (SI Appendix, Fig. S7) as well as in
additional daughter cell lines. Because SNP arrays are not sensi-
tive to distinguish copy number changes greater than two, it is not
surprising that only the fibroblast subclone 2F12 was reported to
have amplification on chr4: 86,288,42386,420,622, as this sample
had a CN = 5 compared with CN = 4 among other lines. Our
finding indicates that at least a subset of putative somatic struc-
tural variations identified in our dataset were inherited from pa-
rental fibroblast cells, although we were not able to unmask very
low frequent CNVs in these cells. We also queried each putative
CNV against the Database of Genomic Variants (dgv.tcag.ca/) to
determine whether these CNVs have been reported previously.
We found that all of the putative de novo CNVs have been pre-
viously reported except for one duplication event (chr1: 23,884,369
23,941,735) in PF1 fibroblast subclone 2F3. Based on these exper-
imental findings and database searches, it is likely that most, if not
all, of the putative CNVs are preexisting variations.
Discussion
In this study, we have taken a unique approach to compare the
mutational history of iPSCs with fibroblast subclones derived
from the same fibroblast population. This experimental design
made it possible to directly assess whether iPSC-reprogramming
process enhances mutagenesis, because the fibroblast subclones
Kwon et al.
were derived similarly to the iPSCs, except for the reprogram-
ming factor treatment. Our data therefore provide strong evi-
dence that iPSC reprogramming is not mutagenic.
Our findings also provide strong support that a large majority of
genetic variations present in iPSCs are preexisting mutations,
which are likely to be present in a small clonal population of the
parental somatic cells (1, 15, 20, 21). These rare mosaic variants
existing in a pool of somatic cells are then overrepresented in a
clonal iPSC line, leading to misclassification as de novo variants.
Therefore, it is still not trivial to distinguish true de novo variants
associated with the reprogramming process from rare mosaic var-
iants inherited from parental somatic tissue.
Recent advances in genomics, especially in single-cell genomics,
have revealed that genome mosaicism in human somatic tissues is
a far more frequent phenomenon than once believed (2226). In
this study, we demonstrated a great degree of variation in genome
mosaicism among different somatic tissue types. However, the
sharing of putative de novo variants among daughter lines was a
surprising finding, as we previously reported that among thousands
of de novo variants discovered by WGS, clonal iPSC lines derived
from primary bone marrow cells contained only unique variants
(1). Reanalysis of our previously published WGS dataset of bone-
marrowderived iPSC lines (accession no. SRA048525) with
Shimmer using the same filtering criteria still found no overlapping
GENETICS
de novo variants among the three iPSC lines (SI Appendix, Fig. S4).
Our findings suggest the possibility that different somatic tissues
may have varying levels of mosaic mutations. Gene set enrichment
analyses showed that genes containing de novo or preexisting
variants did not cluster in any specific functional pathway and there
were no indications that they contributed to increased reproductive
fitness. Therefore, it appears that the de novo and the preexisting
variants were largely randomly occurring mutations.
We observed that the iPSC lines contained more putative new
variants (before deep resequencing to detect their existence in the
parental cells) than the fibroblast subclones (Figs. 2A and 3A). One
possible reason for the increased mutation load in iPSCs is that
iPSCs were collected at later passages compared with fibroblast
subclones, increasing the chance for rare variants to rise in fre-
quency. Because our WES analysis was limited to detect variants
with minor variant frequency >0.001, any variants with a frequency
of <0.001 will remain undetected using our WES approach. Al-
ternatively, the differences in mutation load could simply be caused
by chance, because the total numbers of cell lines were relatively
small, especially for the iPSCs. Such differences may disappear if
more cell lines were analyzed. Finally, the data may reflect true
differences in the numbers of inherited variants between iPSCs and
fibroblast subclones. Therefore, this is an issue that requires ad-
ditional investigation in the future.
The majority of CNVs we detected were also found to be pre-
existing variations that were either present in the parental fibroblast
cells or were previously reported to be present in the general
population (14). We observed one difference in genomic stability of
iPSCs where a chromosome 12 duplication occurred in the highest
passage iPSC line, iPSC3 (n = 39). Several other studies have al-
ready reported high incidence of chromosome 12 duplications in
iPSCs grown in culture for long term and have hypothesized that
these iPSCs undergo high selective pressure during prolonged
in vitro culture (10, 2729).
Interestingly, we identified a mosaic translocation event,
t(5;11)(q35;q13), that has been previously reported to be associ-
ated with acute myeloid leukemia (AML) subtype M2, in one fi-
broblast subclone where the parental fibroblast sample (PF1) was
isolated from a patient with FPD harboring the Y260X mutation
in RUNX1 (17, 30). However, RUNX1 mutations are not known to
affect mutagenesis process or DNA repair, and our data do not
show significant difference between the numbers and types of
mutations between daughter lines of PF1 and those from the
healthy donor (PF2).
PNAS Early Edition | 5 of 6
 Collectively, our study supports the hypothesis that the iPSCs
do not have increased numbers of de novo mutations and that
somatic mutations arise randomly in cells where there is a great
varying degree of genomic variations in different parental
tissue sources.
Materials and Methods
Fibroblast Single-Cell Expansion and iPSC Generation. Clonal populations
arising from single cells were derived by FACS sorting early passage fibro-
blasts (P5) in a 96-well plate and clonal populations were collected at P9 for
gDNA extraction. Two iPSC lines and three iPSC line pairs were derived from
fibroblasts PF1 and PF2, respectively. PF1 was a male patient with FPD (III-5 in
ref. 17) and PF2 samples were derived from a clinically healthy male control
donor (18). Full methods for reprogramming of these iPSC lines are de-
scribed elsewhere (17, 18).
Whole Exome and Custom Targeted Capture Sequencing. The exome capture
was performed according to Illuminas TruSeq Exome Enrichment Kit pro-
tocol. Targeted capture sequencing was performed according to Nim-
blegens SeqCap EZ Choice protocol. Detailed methods are described in SI
Appendix, SI Materials and Methods.
Discovery of Potential Somatic Mutations Using Whole Exome Sequencing.
Whole exome sequence reads for each sample were aligned to the hg19 ref-
erence sequence using novoalign version 2.08.02 and PCR duplicates were
removed using SAMtools (31). We ran Shimmer (19) to perform all-versus-all
pairwise comparisons usingminqual 20 andtestall options, and filtering
mutation predictions with a read depth of >750 in either sample or a q value
of >0.05 as likely artifacts. This resulted in a list of 425 single nucleotide
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changes and 25 indels for PF1 and 357 single nucleotide changes and 13 indels
for PF2 to be interrogated further in the custom capture and sequencing.
Variant Allele Frequency Calculation from Targeted Custom Capture Deep
Sequencing. We aligned sequencing reads derived from the targeted cus-
tom-captured samples to hg19 and reads from multiple libraries were merged
into a single BAM file for each sample. Allele counts for SNVs were computed
from these alignments, including only base calls with phred quality of 20 or
greater, and highest posterior density (HPD) confidence intervals were de-
termined using the CRAN binom package with confidence level of 0.99999
(cran.r-project.org/web/packages/binom/).
Structural Variation Detection and Validation. CNV analysis was performed
using Illuminas Human OmniExpressExome SNP array (958,178 SNPs) on 26
samples, including two fibroblast parental cell lines, 15 fibroblast subclones,
eight hiPSC lines, and one technical duplicate of the PF1 parental fibroblast
line. CNVs for all samples were identified with three independent calling al-
gorithms, PennCNV (32), CNVPartition (Illumina), and Nexus v7 (Biodiscovery).
Unique CNVs were validated using FISH and SKY for large variations (>500 kb)
and ddPCR was used to validate smaller CNVs (SI Appendix, SI Materials and
Methods, Tables S10 and S11, and Figs. S5S8).
ACKNOWLEDGMENTS. We thank Dionyssia Clagett (Georgetown University)
for establishing fibroblast lines from patients with FPD, Ms. Ursula Harper for
performing short tandem repeat mapping for sample identify confirmation,
members of the NIH Intramural Sequencing Center for their support on WES
and targeted sequencing, and Julia Fekecs for expert design of Fig. 1. The
research was supported by the Intramural Research Programs of National
Human Genome Research Institute and National Heart, Lung, and Blood
Institute, NIH.
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