Direct, genome-wide assessment of DNA mutations
in single cells
Michael Gundry, Wenge Li, Shahina Bano Maqbool and Jan Vijg*
Department of Genetics, Albert Einstein College of Medicine, New York, NY 10461, USA
Received July 18, 2011; Revised September 2, 2011; Accepted October 11, 2011
DNA mutations are the inevitable consequences of
errors that arise during replication and repair of DNA
damage. Because of their random and infrequent
of DNA mutations in the genome of somatic cells
has been difficult. Random, low-abundance muta-
tions are currently inaccessible by standard high-
throughput sequencing approaches because they
cannot be distinguished from sequencing errors.
One way to circumvent this problem and simultan-
eously account for the mutational heterogeneity
within tissues is whole genome sequencing of a rep-
resentative number of single cells. Here, we show
Drosophila melanogaster S2 and mouse embry-
onic fibroblast populations after treatment with
the powerful mutagen N-ethyl-N-nitrosourea. This
method can be applied as a direct measure of
exposure to mutagenic agents and for assessing
In spite of the enormous progress in genomics, the field is
still very much focused on averages. Virtually all
sequencing cancer genomes, typically only a very small
fraction of all mutations present in the tumor are
low-abundance mutations present in a limited number of
cells. In principle, such mutations could be detected by
sequencing a large number of times across the same
locus, i.e. at great sequencing depth. However, the high
error rate of current high-throughput sequencing plat-
forms (0.1–1%) effectively
DNA. example, in
mutations (2,3). To account for sequencing errors,
current protocols for mutation detection are based on a
consensus model, i.e. finding the same event in multiple,
independent reads from the same locus. This allows only
the detection of clonally amplified mutations present in
most or all of the cells in a tissue sample and essentially
constrains access to low-abundance mutations. One way
to circumvent this problem is to sequence genomes of in-
dividual cells after whole genome amplification (WGA)
(4). Every mutation in that cell at a particular locus now
acts as the consensus sequence. This is schematically
depicted in Figure 1A.
To demonstrate that the accurate detection and quanti-
fication of somatic mutations in the genomes of single
eukaryotic cells is feasible, we developed a protocol in
which single cells are subjected to WGA followed by
paired-end sequencing. After alignment to a reference
sequence, mutations are detected as differences between
the amplified single-cell genomic DNA and the reference
sequence normalized to the non-amplified genomic DNA
of the mother cell population. The efficacy of this protocol
was validated by determining mutational spectra in single
cells from Drosophila melanogaster (S2 cells) and mouse
embryo’s (mouse embryonic fibroblasts; MEF) exposed to
the powerfulpoint mutagen
MATERIALS AND METHODS
Cells and treatment
The Drosophila S2 cell line was acquired from Invitrogen.
S2 cells were cultured in Schneider’s insect medium
(Sigma) supplemented with 10% FBS and penicillin/
streptomycin (GIBCO) at 29?C. Primary MEFs were col-
lected from LacZ transgenic C57BL/6 mice and cultured
in DMEM (GIBCO), supplemented with 10% FBS, peni-
cillin and streptomycin. S2 cells and MEFs (passage 5)
were treated with 500mg/ml (4.3mM) ENU or solvent
control in serum-free medium for 30min. Following
*To whom correspondence should be addressed. Tel: +718 678 1151; Fax: +718 678 1016; Email: firstname.lastname@example.org
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
Nucleic Acids Research, 2012, Vol. 40, No. 5 Published online 15 November 2011
? The Author(s) 2011. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
treatment, cells were washed three times in PBS and
cultured in serum-supplemented medium for an additional
72h before proceeding with mutation analysis.
Single cell isolation and WGA
Single S2 cells or MEFs were collected under an inverted
microscope by hand-held capillaries, deposited in PCR
tubes along with 1ml PBS, and immediately frozen on
dry ice. The remaining cells were frozen on dry ice and
stored at ?80?C. Multiple displacement amplification was
performed on single cells using the REPLI-g UltraFast
Mini kit (Qiagen, Santa Clara, CA, USA) according to
the manufacturer’s instructions with some modification.
After lysis, an initial 30-min amplification at 30?C was
used, followed by a 23.5-h amplification at 37?C. The
DNA was purified using AMPure XP magnetic beads
(Agencourt, Beverly, MA, USA) and the yield measured
using the NanoDrop 1000 spectrophotometer (Nanodrop
Technologies LLC, Wilmington, DE, USA). Single cell
Figure 1. Somatic mutation detection using single cell sequencing. (A) Somatic mutations in tissues are rare and therefore found only in single
sequencing reads from which they are routinely filtered out as sequencing errors during post-alignment processing. Adopting a single cell approach
overcomes this limitation by transforming each somatic event into a consensus variant call. (B) Schematic depiction of the single cell sequencing
protocol used for Drosophila S2 cells.
Nucleic Acids Research,2012, Vol.40, No. 52033
reactions with yields >1mg were tested for locus dropout
at 10 (S2) or 8 (MEF) loci using real-time PCR. Aliquots
from the single cell reactions and from an unamplified
control sample were diluted to 1ng/ml. Real-time PCR
(StepOne Plus, Applied Biosystems, Foster City, CA,
USA) was performed with Fast SYBR?Green Master
Mix (Applied Biosystems, Foster City, CA, USA) using
2ng of input from each of the diluted samples and a final
primer concentration of 187.5nM. The relative abundance
of each locus was estimated by comparing the Ct values
from the unamplified control and each individual single
cell. Cells with the highest number of loci present at a
sufficient level (relative abundance>0.25) were chosen
for Illumina sequencing.
Sequencing library construction
Up to 5mg of DNA was used as input to make Illumina
libraries (5). Unamplified control DNA was extracted
from the frozen cell culture mixtures isolated after the
Drosophila S2 and MEF treatments. Drosophila S2
unamplified control DNA or single cell DNA was
randomly fragmented using the Covaris S220 instrument
(Covaris, Woburn, MA, USA). The fragmented DNA was
end-repaired and size-selected to 475–525bp using 1.5%
agarose (Certified Molecular Biology Agarose, Bio-Rad,
Hercules, CA, USA). The size-selected material was
A-tailed and ligated to Illumina paired-end adapters
(IDT, Coralville, IA, USA). A second size-selection was
performed under the same conditions to reduce the fre-
quency of chimeras. Approximately 10ng of the product
was used as input for 10 cycles of enrichment PCR with
2U of Platinum Pfx DNA polymerase (Invitrogen,
Carlsbad, CA, USA), 1? Pfx buffer, 2mM MgSO4,
400mM dNTPs and 1mM Illumina enrichment PCR
primers (IDT, Coralville, IA, USA). MEF unamplified
control DNA or single cell DNA was digested with 50U
of MseI (NEB, Ipswich, MA, USA), end-repaired using
5U of Mung Bean Nuclease (NEB, Ipswich, MA, USA)
and size-selected using 1.5% agarose to 250–350bp. The
size-selected material was A-tailed and ligated to Illumina
paired-end adapters. Approximately 10ng of the product
was used as input for 10 cycles of enrichment PCR.
Sequencing and data analysis
sequencing (S2 cells) or 121-bp single-end sequencing
(MEFs) on the HiSeq 2000 (Illumina, San Diego, CA,
USA). Raw sequencing data was aligned to the dm3
(S2 cells) and mm9 (MEFs) reference sequences using
BWA (6) with default parameters. Reads that did not
align uniquely to the genome were discarded to eliminate
false positives due to mapping artifacts. The aligned
sequence data was processed using GATK (7) to realign
reads containing indels or a high entropy of mismatches,
recalibrate the base quality scores and to compute
coverage data and statistics. Somatic point mutations
and germline variation were scored using a pipeline
composed of SAMtools (8) and the VarScan (9) somatic
command. A minimum base quality score of 20 and a
minimum mapping quality score of 20 were set in the
were sequencedusing 108-bp paired-end
VarScan command. For the Drosophila S2 cells, a min-
imum read depth of 20 was required for both the unamp-
lified sample and the single cell along with a minimum
mutant allele frequency of 20% for point mutations
found in the single cell. Additionally, a strand bias script
was also used that filtered out events where the variant
allele was biased towards reads aligning to one strand. For
the MEFs, a minimum read depth of 10 was required for
both the unamplified sample and the single cell, along with
a minimum mutant allele frequency of 40% for point mu-
tations found in the single cell. The VarScan parameters
used for somatic mutation discovery in the S2 cells and
MEFs were found to achieve the appropriate balance
between reducing false positives and genotyping a high
fraction of the genome/target region. More stringent par-
ameters did not lead to a considerable change in the
measured mutation frequency. Somatic events found in
multiple single cells were discarded, as were events found
in at least one read in the unamplified control sample.
Filtered somatic point mutations were visually validated
using a custom IGV (10) batch script that recorded images
of aligned reads at each locus containing a somatic point
mutation. Select point mutations were chosen for further
validation using Sanger sequencing. Primers were designed
to flank either side of the mutant of interest. DNA from
the single cell containing the somatic mutation and the cell
line were tested and the trace images were inspected to
confirm that the wild type and mutant alleles (trace
peaks) were found at the expected ratio.
Mutation spectra and localization
Analysis of the localization and spectra of point mutations
was performed using GATK. Functional classification of
point mutations wasperformed
GenomicAnnotator tool and ANNOVAR (11). Strand
specificity was measured for those mutations falling
within genic regions based on the assumption that
ENU-induced mutagenic lesions only occur at guanine
and thymine bases (12). The expression levels (RPKM
values) of Drosophila genes containing detected point mu-
tations were obtained from an RNA-Seq experiment (13)
with the S2 cell line, and the distribution of these values
was compared to the genome-wide distribution to test for
a correlation. The analysis was repeated for detected point
mutations in MEFs but a microarray data set (14) was
used due to lack of an appropriate RNA-Seq data set.
The proximity of detected point mutations to replication
origins was tested by annotating the single cell point
mutation tracks with high-throughput replication timing
data from the S2 cell line (15) and MEFs (16). A genome
accessibility data set (17) was used to test for a correlation
between point mutation localization and chromatin state
in the S2 cells.
Estimation of the mutation load in ENU-treated and
untreated Drosophila S2 cells
To assess somatic mutation spectra in single cells in a
genome-wide manner we first used S2 cells derived from
2034 Nucleic Acids Research, 2012,Vol.40, No. 5
D. melanogaster, an organism with a genome size of
160MB. The strategy followed is outlined in Figure 1B.
Individual single cells were picked from an S2 cell culture
72-h after treatment with 4.2mM of the powerful
point mutagen ENU or mock-treated with solvent
(control). At 72-h post-treatment virtually no lesions
remain (18) and cell survival is >90% (not shown). Each
single cell was lysed and subjected to WGA using an
optimized multiple displacement amplification (MDA)
protocol (19). Using a qPCR protocol, specifically
designed for the purpose, amplified products for each
cell were first screened to verify successful amplification
by using primer pairs distributed over the different
chromosomes (Supplementary Figure S1). Only samples
showing amplification for all or most tested chromosomes
were further processed to generate sequencing libraries for
the Illumina HiSeq platform. In this way we prepared
libraries of three untreated and three ENU-treated cells.
For comparison, a similar library was made from unamp-
lified total genomic DNA from the untreated S2 cell
To identify all possible mutations, either spontaneously
formed in the unexposed, control cells or induced by ENU
in the treated cells, the libraries were sequenced using a
paired-end module. Variant analysis revealed point muta-
tions, small indels and genome rearrangements. Since
ENU is a point mutagen our subsequent analysis
was based on this type of mutation only. Aligned
sequence data from the unamplified sample and the
MDA-amplified single cells were compared and all differ-
ences with the reference sequence were recorded as
variants. Variants with sufficient coverage (20?) in both
the unamplified and the single cell sample were classified
as ‘germline’ or ‘somatic’ based on whether the variant
was shared between the unamplified sample and the
single cell. Somatic variants were further processed using
a strand-bias filter and visually validated using the
Integrative Genomics Viewer (IGV).
The results indicated sufficient coverage (20?) for
genotyping at between 40% and 80% of target bases
(Table 1). The incomplete coverage is due to amplification
bias, which can be pronounced especially with small
template amounts (19). While we optimized the MDA
protocol, locus dropout was still observed, as was a sig-
nificant level of allele dropout. The latter was measured
using both heterozygous SNPs present in our S2 cell line
population and the mutant read frequency distribution,
which produced similar results. A comparison of the
mutation load in the control and ENU-treated cells
showed a 7.5-fold induction of point mutations by ENU
on average in the exposed cells (Figure 2A, Table 1).
We chose multiple somatic mutations for validation with
Sanger sequencing using the remaining amplified material
from our single cells and found no evidence of false posi-
tives (for an example, see Supplementary Figure S2).
While spontaneous mutations present in the untreated
cells could be expected to occasionally be homozygous, all
ENU-induced mutations are likely to involve one allele
only. The S2 cell line is known to be tetraploid (13)
(Supplementary Figure S3) and assuming an equal repre-
sentation of each allele in the whole genome-amplified
material from our single cells, we would expect a quarter
of the reads aligning across a spontaneous or induced
mutation to contain the mutant base. Figure 2B shows
the mutant read frequencies across chr2L for the
ENU-induced mutations. While we did find the expected
peak read frequency of 25% for chr2L, the significant tail
to higher frequencies indicates the unequal amplification
across the four alleles. Since the S2 cell line is male, there
are two X chromosomes in addition to the four sets of
autosomes. Hence, we would expect a read frequency
peak at 50% rather than 25% for chrX and this is
indeed what was found (Figure 2B).
Estimation of the mutation load in ENU-treated and
To apply the same strategy to mammalian cells is signifi-
cantly more expensive because of the larger genome size.
Therefore, we slightly modified the procedure shown in
Figure 1B, applying a reduced representation approach.
For this purpose we used mouse embryonic fibroblast
(MEF) populations, either treated with 4.2mM ENU or
mock-treated with solvent only. Instead of preparing
sequencing libraries directly using randomly fragmented
DNA, we digested whole genome-amplified DNA from
two treated and two control MEFs as well as unamplified
genomic DNA from the MEF population with MseI, a
four-base cutter with a TTAA cleavage site.
Following digestion, a fragment size range (250–350bp)
corresponding to a target region of ?300MB was isolated
Table 1. Single cell sequencing data
Single cell Point
Bases in genome with
sufficient coverage (MB)
target region (%)
S2 Cont. 1
S2 Cont. 2
S2 Cont. 3
S2 ENU 1
S2 ENU 2
S2 ENU 3
MEF Cont. 1
MEF Cont. 2
MEF ENU 1
MEF ENU 2
Nucleic Acids Research,2012, Vol.40, No. 52035
from agarose gels and sequenced using 121-bp single-end
reads. The processed data revealed a significant number of
point mutations induced by ENU in the two cells from the
exposed population, similar to what was observed for the
S2 cells (Table 1). Due to the nature of the reduced rep-
resentation library, the strand bias filter could not be used
and therefore a more stringent mutant read frequency
cutoff (>40%) was adopted. The results indicate a
35-fold induction of point mutations in the ENU-treated
MEFs. Previously, using a lacZ reporter in the same
cell population, a significantly smaller number of ENU-
induced mutations was observed (20), underscoring the
reduced sensitivity of reporter systems, which can only
detect mutations that alter the phenotype to a consider-
able extent (21).
The much higher fold induction of mutations in the
ENU-treated MEFs than in the S2 cells is almost
entirely due to a lower baseline mutation frequency in
the two untreated MEFs. This is not surprising since the
S2 cell line used has a long history of passaging during
which mutations are likely to have accumulated. Baseline
levels of somatic mutation frequencies are obviously very
difficult to determine with high accuracy and in this case
also depend on the cut-offs used to filter out potential
artifacts. Here, we were more interested in comparing
the absolute number of mutations per MB induced
by ENU in cells from the two species, which proved
remarkably similar. Indeed, the ENU-induced mutation
frequency in the MEFs was only 30% higher than
that found in the S2 cells (Table 1). Somatic mutation
frequencies are a measure for the efficiency of an
organism to cope with DNA damage and it is somewhat
surprising that cells from such disparate species are very
similar in this respect. More extensive studies with differ-
ent, freshly isolated cell types from these species should
provide a definite answer.
Cross species comparison of the spectra and localization
of ENU-induced and spontaneous mutations
A major advantage of direct sequencing is that the
mutation spectra can immediately be visualized across
the genome. The ENU-induced spectrum was highly
Number of mutations
Percentage of reads
containing mutant allele (%)
Number of mutations
Percentage of reads
containing mutant allele (%)
Figure 2. Observed somatic point mutations. (A) Genome-wide sequence coverage and mutation localization in an untreated cell and an ENU-
treated cell. The outer track represents binned coverage with an upper cutoff of 50?. The inner track shows the location of detected point mutations
(represented as dark points). (B) Histograms of mutant read frequencies for point mutations on chr2L and chrX. The dotted lines indicate a normal
distribution with mean of 25 for chr2L and 50 for chrX with standard deviations of 22.
2036Nucleic Acids Research, 2012,Vol.40, No. 5
consistent across individual cells from the same popula-
tion, but a larger fraction of C:G!T:A mutations was
found in the S2 cells (Figure 3A). This may be due to a
more efficient repair of O6-ethyl-guanine adducts by the
(Mgmt) compared with its Drosophila homologue. In
spite of this difference, the similarity between the two
species also at this level is striking. The spontaneous
mutation spectra observed in the untreated cells showed
considerably lower levels of A:T!T:A mutations, which
are known to be highly enriched following treatment with
alkylating agents (12,22).
Since ENU is a small, direct-acting agent, we did not
expect to see a large bias for mutations localized in
accessible regions of the genome. By comparing data on
the accessibility of the S2 cell line with the coordinates of
our point mutations, we determined that there was no
correlation between mutation localization and genome ac-
cessibility. There was also no correlation in either the
MEFs or S2 cells between the mutation frequency and
proximity to a replication origin or localization in a func-
tional category (exon, intron or intergenic region).
Analysis of the ENU-induced mutations falling within
genic regions in the two MEF cells showed evidence of
transcription-coupled repair, with a lower fraction of
ENU-induced mutations occurring at T and G bases,
the predominant adduct bases, on the transcribed strand
than the non-transcribed strand (P<0.01) (Figure 3B).
S2 SC #1
S2 SC #2
S2 SC #3
MEF SC #1
MEF SC #2
Fraction of mutations
G->A G->C G->T T->A T->C T->G All
Somatic point mutations
G->A G->C G->T T->A T->C T->G All
Somatic point mutations
Figure 3. Somatic point mutation spectra and localization. (A) Mutation spectra for the control and ENU-treated S2 and MEF single cells. Due to a
limited number of point mutations, the control cells were combined for the analysis. (B) Strand of origin for ENU-induced mutations found in genic
regions of MEFs. (C) Strand of origin for ENU-induced mutations found in genic regions of S2 cells.
Nucleic Acids Research,2012, Vol.40, No. 52037
This bias appeared strongest for TA transversions
(P<0.0002), supporting previous results at the endogen-
ous HPRT gene locus (22). No evidence for any
transcription-coupled repair process (Figure 3C) was
seen in the S2 cells, in keeping with results obtained with
Drosophila Kc cells (23) and the absence of homologues
of either CSA or CSB, the main TCR genes, in Drosophila
In the present work, we addressed the problem as to how
low-abundant DNA mutations, for example, as typically
induced by environmental mutagens, can be detected
directly using ultra-high-throughput sequencing. In prin-
ciple, such low abundant mutations can be detected by
single-molecule sequencing (25). However, this necessarily
involves the sequencing of fragments derived from differ-
ent cells and precludes insight into the mosaic basis of
somatic mutations in tissues or cell populations. Indeed,
the absence of robust methods for analyzing genomes
at the level of the single cell essentially constrains access
to the complexity of biological tissues (26). For example,
cancerous tissue is notoriously heterogeneous with each
cell carrying its own unique capabilities for growing into
a full-blown tumor (1). The ability to analyze subclonal
genetic diversity will greatly expand the accessible clinical
information about a particular cancer in a particular
Another area of application of single-cell genome
sequencing is in monitoring stem cells. The genomic integ-
rity of human embryonic stem cells and induced pluripo-
tent stem cells has been questioned due to a high observed
rate of point mutations (28), copy number variations (29)
and changes in genome-wide CpG methylation (30). These
studies identified potentially hazardous somatic muta-
tions/epi-mutations that had clonally expanded through
the population. However, there may also be low-
abundance mutations present in the stem cell populations
missed by the aforementioned analyses that need to be
addressed before clinical use can be considered.
Finally, during development, maturation and aging the
genome of somatic cells is subject to the continuous oc-
currence of random genome sequence alterations, which
gradually diminish intra-organ homogeneity and may lead
to loss of coordination of expression among multiple
genes in functional pathways or networks (31). Such
emerging cell-to-cell variability is only amenable through
single-cell genomics approaches.
In this study, we have taken the first step towards com-
prehensively analyzing mammalian single-cell genomes
using next-generation sequencing.
evidence of an induced mutation load in single ENU-
treated cells from a Drosophila cell line. The high level
of induction and the consistency of the results across the
three ENU-treated cells provide strong evidence that the
experimental results for the ENU-induced cells are
accurate. Next, we developed a reduced-representation
assay to repeatthe experiment
observed a consistent mutation frequency across the two
We first showed
using MEFs. We
single ENU-treated MEFs, similar to the levels found in
the treated S2 cells. The mutation frequencies observed in
the treated MEFs were two-fold higher than those previ-
ously estimated using a lacZ transgenic reporter gene (20).
The discrepancy can be explained, at least in part, by the
fact that the reporter gene cannot detect mutations that do
not inactivate the b-galactosidase enzymatic activity (21).
While ourresults conclusively
mutation frequencies in both S2 cells and MEFs after
exposure to ENU, our data does not allow for accurate
estimates of the frequency of spontaneous mutations in
control cells. For the control MEF cells, it is possible to
makea comparison with
frequencies observed with our lacZ reporter system in
these same cells (32). While we do find higher mutation
frequencies by direct sequencing than with the reporter
system (in keeping with the inability of the lacZ positive
selection system to detect silent mutations), they are in the
Background mutation frequencies are equal to the sum
of the spontaneous mutation frequencies within the indi-
vidual cells and the background error rate of the assay due
to mutations introduced during the WGA step. An error
introduced early in MDA would be found in 12.5% of the
sequences in a diploid cell and 6.25% of the sequences in a
tetraploid cell on average (25). The kinetics of the ampli-
fication process, i.e. the fact that multiple polymerase mol-
ecules may be operating on the original template strand
when the initial error is introduced, may further reduce the
probability of significant errors occurring. The percent-
ages listed above are averages however, and it is possible
that an error produced early in MDA could be randomly
selected for, leading to a false positive call present in the
majority of reads aligning at a locus. Additionally, the
high degree of allele dropout observed in many of our
samples increases the probability that some artifacts
produced early in MDA could be found in a significant
proportion of reads aligning at a locus. In order to obtain
a more quantitative estimate of the spontaneous mutation
frequency, more extensive studies are needed, using
maximum-likelihood methods for estimating mutation
frequencies from the high-throughput sequencing data,
for example, as described by Lynch (33).
The mutant spectra observed in the treated S2 and MEF
cells agrees with data obtained using reporter genes
(34–36), providing additional evidence for the accuracy
of our measured ENU-induced mutation loads. A
common argument against the use of reporter genes is
that they may not be representative of genome-wide
events, due to both sequence specificity and their depend-
ence on a phenotypic change. ENU is a small direct-acting
agent with a lack of sequence specificity and, therefore,
it was not surprising that no major differences were
found between reporter gene data and our genome-wide
While this analysis was limited to point mutations, the
same methodology can be applied to investigate small in-
sertions and deletions (InDels) and structural variation.
Indeed, the paired-end sequencing approach allows us to
detect structural alterations as invalid alignments to the
reference genome sequence (37). If the mapped locations
2038 Nucleic Acids Research, 2012,Vol.40, No. 5
of the ends of a paired-read have abnormal distances,
orientation or chromosomal localization, then a genomic
rearrangement is suggested. While we were clearly able to
detect such events, ENU is a point mutagen and we did
not find an increase of genome rearrangements in the
treated cells (not shown).
In summary, these results show for the first time how
massively parallel sequencing can be used effectively for
measuring random, low-abundance mutations in somatic
cells. This opens up the possibility to analyze intra-tissue
heterogeneity of cellular genotypes. Importantly, our
methodology provides a direct, comprehensive approach
for estimating an individual’s risk from exposure to mu-
tagenic agents, such as radiation.
Supplementary Data are available at NAR online:
Supplementary Figures S1–S3.
The authors acknowledge Donald Conrad, Alex Maslov,
Brent Calder and Silvia Gravina for many helpful discus-
sions of the data and Francesca Faggioli for karyotyping
the S2 cell line.
National Institutes of Health (NIH) (grant numbers
R21ES019520, U54RR024346). Funding for open access
Conflict of interest statement. None declared.
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