Sequencing newly replicated DNA reveals widespread
plasticity in human replication timing
R. Scott Hansena,1, Sean Thomasb, Richard Sandstromb, Theresa K. Canfieldb, Robert E. Thurmanb, Molly Weaverb,
Michael O. Dorschnerb, Stanley M. Gartlera,b, and John A. Stamatoyannopoulosb,1
aDepartment of Medicine, Division of Medical Genetics, andbDepartment of Genome Sciences, University of Washington School of Medicine, Seattle,
Contributed by Stanley M. Gartler, and approved November 4, 2009 (received for review September 24, 2009)
Faithful transmission of genetic material to daughter cells involves a
characteristic temporal order of DNA replication, which may play a
genome-scale approach—Repli Seq—to map temporally ordered rep-
licating DNA using massively parallel sequencing and applied it to
human cell types. The method requires as few as 8,000 cytometry-
fractionated cells for a single analysis, and provides high-resolution
DNA replication patterns with respect to both cell-cycle time and ge-
nomic position. We find that different cell types exhibit characteristic
replication signatures that reveal striking plasticity in regional replica-
tion time patterns covering at least 50% of the human genome. We
also identified autosomal regions with marked biphasic replication
timing that include known regions of monoallelic expression as well
as many previously uncharacterized domains. Comparison with high-
resolution genome-wide profiles of DNaseI sensitivity revealed that
comprising clustered DNaseI hypersensitive sites, and that replica-
tion time is better correlated with chromatin accessibility than with
gene expression. The data collectively provide a unique, genome-
wide picture of the epigenetic compartmentalization of the human
genome and suggest that cell-lineage specification involves exten-
sive reprogramming of replication timing patterns.
chromatin structure|gene expression|tissue specificity
cells grow, replicate their chromosomes, and segregate the
duplicated genome to their daughter cells. DNA replication is
central to this process, and occurs by a complex series of events
usuallyina defined sequential order (1).The moleculardetails and
the mechanisms establishing and maintaining the replication pro-
the human replication program varies between different cell types,
and accessibility of human chromatin.
Beginning with the experiments of Taylor et al. using tritiated
thymidine for visualizing DNA replication (2), nucleotide in-
corporationstudies haveestablishedthat replicationinanimalcells
is organized into discrete zones of similar replication timing con-
sisting of multiple replication origins and their associated replicons
that vary in size from 30 to 450 kb (1, 3). Multireplicon zones of
visualized in metaphase chromosomes as heritable “replication
bands” that generally correspond to classical cytogenetic Giemsa
be visualized in interphase cells as stable structures with defined
subnuclear positioning that is highly heritable in daughter cells (5).
Like classical banding, replication banding appears to be quite
replication program. Some large-scale molecular studies of differ-
ent human cell types appear to confirm the basic similarity of the
replication program across developmental lineages (6–9).
he eukaryotic cell cycle consists of an orderly process in which
In contrast to these stereotypical patterns, localized plasticity
in the human replication program has been described in the
context of several cell-type- or developmental stage-specific
genes, where earlier replication is associated with expression and
later replication with repression (1, 10–13). More recently, ge-
nome-wide studies in both mouse and Drosophila have observed
replication time plasticity over about 20% of the genome (14,
15). The regions differing in replication time between cell types
include those with a subset of genes showing the expected pat-
tern of early replication in expressed cells and later replication in
repressed cells, as well as those lacking expression differences.
DNA replication has also been linked to other epigenetic fea-
both mouse and Drosophila, early replication is more closely as-
sociated with histone acetylation than with gene expression per se
(14, 15), although replication origin efficiency has been suggested
to be more dependent on transcription than on open chromatin
(16). The DNA methylation-replication time connection comes
from observations of demethylation-induced advancement of
replication commonly seen at a number of loci, including the in-
active X chromosome (12); advanced replication time is also as-
sociated with abnormal hypomethylation seen in ICF syndrome
cells deficient in the DNMT3B methyltransferase (13, 17).
Although it is clear that replication timing, DNA methylation,
and chromatin modification are important epigenetic factors
with respect to transcription, their relative importance and
interdependence have yet to be fully elucidated. For example,
the fully methylated HPRT gene on the inactive X can undergo a
spontaneous advance in replication that is not associated with
transcription, although demethylation-induced reactivation oc-
curs at a much higher frequency in the advanced state (12).
Similarly, escape from X inactivation in ICF syndrome cells for
the abnormally unmethylated G6PD gene only occurs if repli-
cation is advanced toward an active-X-like pattern (13).
isa criticalepigenetic componentofcellular function inaddition to
its role in the faithful replication of DNA sequence information
(18). Toensurethe transmissionofcellular identity and function, it
Author contributions: R.S.H., T.K.C., M.O.D., S.M.G., and J.A.S. designed research; R.S.H., T.
K.C., and M.W. performed research; R.S.H., S.T., R.S., R.E.T., and J.A.S. contributed new
reagents/analytic tools; R.S.H., S.T., R.S., R.E.T., S.M.G., and J.A.S. analyzed data; and R.S.
H., S.M.G., and J.A.S. wrote the paper.
The authors declare no conflict of interest.
Freely available online through the PNAS open access option.
Data deposition: The RNA levels determined by Affymetrix Exon array analysis are
available from the NCBI GEO database under accession numbers GSM472898,
GSM472903, GSM472910, GSM472944, and GSM472945. The digital DNaseI chromatin
accessibility data are available as released from the ENCODE Project through the UCSC
Genome Browser at http://genome.ucsc.edu (subId = 295 and subId = 106, narrow peak
data). Newly replicated DNA sequencing data are deposited to the NCBI Short Read
Archive under the Study accession number SRP001393.1.
1To whom correspondence may be addressed. E-mail: email@example.com or
This article contains supporting information online at www.pnas.org/cgi/content/full/
| January 5, 2010
| vol. 107
| no. 1
is evident that the replication-associated inheritance of epigenetic
templates must be faithfully copied as well as the sequence. The
presence at the replication fork of chromatin assembly factors,
histone-modifying enzymes, and DNA methyltransferases is con-
sistent with this function (18). Additionally, the fidelity of DNA
replication itself appears to be influenced by time of replication,
because late domains exhibit increased mutation frequency (19).
To define comprehensively the DNA replication program in
human cells and explore its importance for genome organization
and higher-order transcriptional regulation, we developed a high-
throughput approach for quantifying DNA replication time as a
function of genome position using massively parallel sequencing
(Repli-Seq) and applied it to define the “replicomes” of multiple
human cell lineages including undifferentiated embryonic stem
cells (hESCs). Our analysis shows that a large fraction of the hu-
man genome—at least 50%—exhibits tissue-specific variation in
DNA replication time. Additionally, we find several genomic re-
gions with biphasic patterns that are compatible with allelic var-
iation in replication timing. We also examined the correlation
between replication time and either tissue-specific gene ex-
pression, and chromatin accessibility, revealing strong association
with the latter.
Results and Discussion
Whole-Genome Profiling of DNA Replication Timing by Repli-Seq. To
explore the human replication timing program in different cell lin-
eages, we developed an approach—Repli-Seq—that couples next-
generation sequencing with a well-validated strategy originally
developed for use with sequence-tagged site (STS) markers (20, 21)
(Fig. 1). We labeled newly synthesized DNA strands in vivo with a
pulse of 5-bromo-2-deoxyuridine (BrdU), which is incorporated by
We then sorted labeled cells into six fractions that span all of the
DNA synthesis phase of cell division (G1b, S1, S2, S3, S4, and G2;
Fig. 1A). We isolated the BrdU-labeled nascent strands (BrdU-
DNA) using an anti-BrdU monoclonal antibody, and assembled the
isolated BrdU-DNA fractions into separate Illumina sequencing li-
per replication time fraction, we obtained about 3 million high-
sequence. To produce a visual profile of the progress of DNA rep-
lication along the genome, we computed the normalized density of
mapped tags for each cell-cycle fraction (sequence tag summaries
are given in Table S1). Fig. 1B illustrates the resulting data from a 3
Mb region of chromosome 12 profiled in a B-lymphoblastoid cell
line (LCL; GM06990). Replication in this region initiates very early
flanking regions that contain repressed genes (SOX5 and IFLTD1).
Validation of Repli-Seq with PCR-Based Replication Timing. To con-
applied both approaches to examine the replication timing of the
humanβ-globinlocusonchromosome 11 inB-lymphoblastoidcells
(in which the β-globin locus is silent) and K562 erythroid cells (in
which the locus is active) (Fig. 2). This analysis revealed a tight
Seq. STS markers in the β-globin locus control region (LCR) and a
surrounding region of about 2 Mb replicate during mid- to late-S
phase in lymphoblastoid cells, whereas markers in the far up- and
densities paralleled the STS patterns and provided compatible
replication timing patterns for the inter-STS regions (Fig. 2B). In
K562 cells the β-globin locus replicates in the very early cell-cycle
fraction, G1b, consistent with previous BrdU-based replication
surrounding region extending ±1 Mb that replicates late in non-
globin-expressing cells is early-replicating in K562 cells (Fig. 2,
shaded region). K562 and LCLs display similar patterns of early
replication in the far-flanking regions. Interestingly, the peak of
early replication at the globin gene cluster in K562 corresponds to
the preferred replication origin locations previously identified near
the HBB globin gene (1).
Whole-Genome Replication Time Profiles Across Multiple Cell
Lineages. To identify systematically regions that differ in repli-
cation timing between distinct human cell types genome-wide, we
dermal fibroblasts (BJ), hESCs (BG02), and a third LCL (TL010).
We also confirmed the high reproducibility of Repli-Seq by reap-
plying it to fibroblasts from a new frozen stock (r = 0.98; Fig. S1).
We observed each cell type to display a unique whole-genome
behavior across all cell types (“constant” domains) in combination
with domains manifesting both quantitative and qualitative cell-
specific patterns (“plastic” domains; Fig. 3; Fig. S2). We also iden-
tified several stereotypical patterns of regional replication timing.
that exhibit cell-type-specific early replication flanked by sym-
late replication, as well as more complex patterns (Fig. 3C).
To characterize and compare replication patterns across cell
types genome-wide, we simplified the data for each cell type by
DNA as a function of genomic position. We then analyzed all
pairwise combinations between cell types to ascertain domains of
significant replication time variability, excluding regions with very
low sequence coverage (see Methods). In contrast to the striking
concordance of replication timing in the three different LCLs
and sorted into different fractions of the cell cycle according to DNA content as
shown for this normallymphoblastoid cell line (LCL).Fractionation is continuous
DNA is made into sequencing libraries and sequenced on the Illumina platform,
and the sequence tags are mapped to the hg18 reference genome. (B) Visual-
ization of replicationpatterns asexemplifiedat the LRMP locus in theGM06990
LCL. Mapped sequence tags in this region of chromosome 12 reveal a very early
peak of replication at the lymphoid-specific LRMP gene. Sequence tag densities
for each cell-cycle fraction (below) were calculated over 50-kb windows, nor-
malized according to their genome-wide sequence tag counts, and further nor-
malized at each genomic position by calculating the percentage of total
replication (to avoid biases due to nucleotide composition or mapability).
Repli-Seq approach. (A) Cell-cycle fractionation of newly synthesized
| www.pnas.org/cgi/doi/10.1073/pnas.0912402107Hansen et al.
studied (r ≥ 0.88 for whole-genome comparisons), we found a
surprisinglylarge fraction ofthehuman genome—49%—to exhibit
replication timing plasticity when four different cell lineages were
compared (Tables S2 and S3). Moreover, it is likely that this is a
lower-limit estimate, as inclusion of additional cell types could
substantially increase the spectrum of observed variability.
Replication Time and Genomic Features. We next compared repli-
cation timing patterns with major genomic annotations (Fig. 4;
Table S3). Regions of constant early replication are significantly
(P < 0.05) associated with high gene density, gene expression,
Alu density, GC content, CpG density, and vertebrate nonexonic
conservation. Constant early regions are also significantly de-
pleted of L1 LINEs and satellite sequences, whereas constant
late regions are enriched in these features (Fig. 4; Table S3).
Many of these genomic features are variable in regions exhib-
iting replication plasticity; thus, their patterns of overall enrich-
ment or depletion are generally more modest than those in the
replication constant categories. The gene content in plastic
regions, for example, is depleted by 30% compared to the rest of
the genome, whereas constant early regions have an enrichment of
135% and constant late have a depletion of 83% (Fig. 4; Table S3).
Overall, the plastic region enrichment patterns resemble constant
late domains to some extent by having lower levels of genes, CpG
islands, and Alus, yet they also resemble constant early domains by
being depleted for satellite sequences and L1 LINEs.
Replication Time, Gene Expression, and Chromatin Structure. To
compare replication timing with gene expression, we measured
RNA levels in each cell type using Affymetrix tiling arrays tar-
geting 1.5 million known and predicted exons (see Methods). We
found only a moderate correlation between early replication and
transcriptional output (r = 0.40). We next examined transcrip-
tional output specifically in regions of replication time plasticity
and found that they follow the expected classic pattern of late
replication when repressed and early replication when expressed
(Fig. S3). On a global level, however, these results suggest that
gene expression is largely a surrogate marker for replication time
and that other factors such as chromatin structure may be more
closely or indeed causally related to replication timing patterns.
We therefore examined replication time patterns in relation to
chromatin and nuclear structure.
Association with the nuclear lamina appears to be a physical
marker of repressed genomic domains, and regions displaying
this association have recently been defined in human fibroblasts
(23). We compared these regions with fibroblast replication
atspecificgenomiclocations inthedistal portion of11p15.4 forerythroidand
define a 2-Mb domain (shaded) containing the β-globin gene cluster that is
early-replicating in erythroid cells that express globin genes (K562) and late-
replicating in lymphoid cells where globin genes are silent (GM08729). (B)
Sequence-based replication timing. The Repli-Seq whole-genome sequence-
based data closely match the replication patterns found for specific STSs, but
provide more comprehensive coverage of this 3-Mb region.
Validation of Repli-Seq with STS-based replication timing. (A) Repli-
timing profiles from four cell types across chromosome 4, illustrating unique
lineage patterns. (B) Lineage-specific early-replication patterns. Cell-lineage-
specific early patterns are highlighted in expanded chromosome 4 subregions.
Thelymphoid-specific CENTD1 gene in the 34.6–37.1 Mb regionis atthe apexof
an early-replication peak in the GM06990 LCL, whereas this region is uniformly
expanded regions: LPHN3 in the 60.9–63.4 Mb region (hESC-specific), GYPA-
GYPB-GYPE in the 143.8–146.3 Mb region (erythroid-specific), and PDGFC in the
156.7–159.2 region (fibroblast-specific). (C) Stereotypical replication timing pat-
terns. Shown are major patterns of DNA replication timing observed across the
genome, including (i) regions of constant early replication across cell lineages;
(ii) regions of constant late replication; (iii) regions with cell-specific early repli-
others early); and (v)complexpatterns that vary considerablybetweenlineages.
Cell-lineage-specific replication timing. (A) Comparison of replication
Hansen et al.PNAS
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timing patterns and observed a striking correlation between sites
of nuclear lamina association and regions displaying late repli-
cation (Fig. 4). This observation suggests that late-replicating
regions generically define not only a repressed but also a physi-
cally segregated nuclear compartment.
To examine the relationship between replication timing and
measurements obtained from the same BJ fibroblast and K562 cell
0.64 versus 0.40 over chromosome 11, for example). Indeed, we
lineage-specific differences in RNA levels, although with clearly
compatible alterations in chromatin accessibility (Fig. S4).
Very Early Replication and Replication Initiation Zones. A distinctive
feature of the replication time profiles is the presence throughout
the genome of hundreds of early-replicating regions flanked by
symmetrical, monotonic early-to-late transitions (inverted-Vs in
and apparently represent replication initiation zones together
with associated bidirectional replication forks that travel the en-
tire length of S phase (14, 24, 25). Because the inverted-V apex
regions and other strong G1 replication zones should be enriched
in constitutive and cell-type-specific replication origins, we cata-
loged these segments for hESCs, LCLs, and fibroblasts using
conservative criteria (see Methods). We identified 1131 such G1
regions in H0287 LCLs, 1199 in GM06990 LCLs, 1809 in BG02
hESCs, and 1547 in BJ fibroblasts (Table S4).
Few human replication origins have been fully described with
high confidence, but a recent HeLa cell study identified 266 rep-
lication origins in ENCODE regions (16); 190 of these (71%)
overlap with our combined early-replicating G1 segments, sug-
comparisons between the different cell lines indicated that only
any two cell types, whereas 85% are shared between two LCLs.
Such lineage-specific variability is consistent with the replication
time plasticity analysis reported above and suggests that early-
replication origin firing is highly variable with respect to cell type.
Biphasic Replication Timing. Among the replication timing regions
discovered to have plastic replication is a class of biphasic repli-
cation domains. Many such regions are seen along the X chro-
identify biphasic regions in all cell types, we devised an automatic
search method based on finding low signals in one cell-cycle frac-
tion that are interposed between strong signals of both earlier and
laterreplication (see Methods). Using this approach,we identified
many regions exhibiting biphasic replication (Table S5), although
the regions flagged account for only a small portion of the meas-
ured genome (0.32%; Tables S3 and S5). Included among these is
the imprinted domain associated with Prader-Willi syndrome at
the SNRPN-SNURF locus on chromosome 15, as well as many loci
on the X chromosome in the female LCL (GM06990) that would
be expected to derivefrom the replication differencesbetween the
active and inactive Xs. Interestingly, the majority of biphasic do-
mains appear to have genomic features similar to the pan-lineage/
constant early regions (Table S3).
Biphasic replicationzones on theX chromosome in femalecells
the classic pattern of allelic asynchrony due to X chromosome
inactivation (12, 26). Certain imprinted domains would also be
expected to exhibit replication asynchrony. The extreme biphasic
15 (G1 and G2) was specific to hESCs (Fig. 5B), although the re-
gion exhibits imprinted gene expression in most cell types (27),
including LCLs and fibroblasts, where replication asynchrony at
to ascertain (28). The extreme asynchrony in hESCs is apparently
related to hESC-specific high expression within the region that
includes SNRPN-SNURF transcription and transcription of more
than 70 genes encoding C/D box snoRNAs (Fig. 5B).
Because hESCs are known to exhibit imprinted SNRPN ex-
pression (29), the G1-replicating portion should correspond to
the expressed paternal allele whereas the G2 portion should
correspond to the repressed maternal allele, similar to the order
of allele-specific replication timing in LCLs (28). This allelic
asynchrony was confirmed using an STS-based replication assay
that interrogates a polymorphic BstUI restriction site (28). The
early-replicating DNA component was exclusively the expressed
BstUI-cut allele, whereas the late-replicating DNA component
was exclusively the silent BstUI-uncut allele (Fig. 5C).
Several other biphasic replication regions we identified were
previously recognized as asynchronous, or were candidates for
and a region on chromosome 2 containing the LRRTM1 gene that
appears to be imprinted with a variable pattern of maternal down-
regulation (31). Additionally, 4 of 18 regions previously identified
to replicate asynchronously in LCLs and fibroblasts (32, 33) are
within 1 Mb of a biphasic replication locus (Table S5). One such
region is near IL12B and contains the EBF1 gene that was recently
identified as exhibiting monoallelic expression in cloned LCLs (34).
Major annotated genomic features across a 50 Mb portion of the long arm of
and gene density. (B) Total RNA output measured by Affymetrix exon arrays
across four cell types. (C) Constant early, constant late, and plastic replication
(E) Regions of nuclear lamina association recently reported for human fibro-
blasts (23) correlate well with the regions of late replication we found in BJ
fibroblasts. (F) Chromatin accessibility and density of DNaseI-hypersensitive
sites in BJ fibroblasts. Note the extremely close correspondence with early-
replicating regions in fibroblasts (D above, lower track), and the inverse re-
lationship with lamin-associated late-replicating regions.
Replication timing patterns versus annotated genomic features. (A)
| www.pnas.org/cgi/doi/10.1073/pnas.0912402107Hansen et al.
Aside from such examples, however, we observed a poor corre-
with monoallelic expression. Of 371 genes exhibiting random mon-
oallelic expression in LCL studies (33, 34), only three—PIP3E, TI-
RAP, and XYLT1—overlap a biphasic replication zone identified in
H0287 and GM06990 LCLs. These findings ostensibly suggest that
random monoallelic expression need not significantly perturb repli-
cation time patterns and, conversely, that allelic variation in repli-
Our analysis of biphasic regions only flagged 0.3% of the meas-
ured genome, whereas previous whole-genome studies of repli-
cation time in HeLa cells (35, 36) and mouse leukemia cells (25)
have suggested that 9–20% of these genomes replicate asyn-
chronously. The greater stringency of our definition of asynchrony
may partially explain this discrepancy, but relaxation of our criteria
is unlikely to significantly close the gap based on our preliminary
analyses and visual inspection of the whole-genome replication
between mouse and human may also explain some of this dis-
cordance. Additionally, the previous studies use lower-resolution
cell-cycle fractionation,and cell synchrony also appearstobe lower
than attained by our cytometry-based method and this could result
in the erroneous appearance of asynchrony for some loci at the
extremes of the replication program.
Perspective. In summary, we developed an approach for quanti-
fying human DNA replication time at high-resolution genome-
wide and applied it across multiple human cell types. Even when
performed on a limited range of cell types, the resulting data
reveal a surprising plasticity in human replication programs.
Repli-Seq is readily extensible to additional cell types as well as
to diverse eukaryotic organisms. Because of its low cell-input
requirements (conservatively about 5000 cell equivalents per
replication time point, ≈1000-fold lower than for a typical his-
tone modification), the method is uniquely suited to studying
rare cell populations such as finely differentiated or developing
Direct sequencing of cell-cycle-ordered replication fractions
has several important advantages over array-based approaches
(9, 14, 15). Whereas array-based approaches are confounded by
repetitive regions, massively parallel sequencing with 27–36 bp
read lengths has the potential to interrogate up to 90% of the raw
human genome sequence. Profiling of multiple temporally dis-
tinct replicating fractions coupled with a sequencing-based ap-
proach enables ascertainment of biphasic replication timing that
is difficult or impossible to attain with any methods employing
early- versus late-replication ratios. Continuing improvements in
the throughput of DNA sequencing, combined with the ability to
multiplex samples using barcoding, provide Repli-Seq with con-
siderable cost advantages over other approaches.
Our studies of replication timing and gene expression in
multiple lineages and DNaseI sensitivity in erythroid cells and
fibroblasts suggest that replication time reflects an important
component of the epigenetic compartmentalization of the
genome. These data serve as a basis for further exploration of
cell-type-specific replication programs, their evolutionary con-
servation, their variation in different genetic backgrounds, and
their potential alteration in disease and disease susceptibility.
Materials and Methods
Expanded experimental procedures are provided in SI Methods.
Cell Culture and RNA Analysis, and the Repli-Seq Procedure. Allcellsweregrown
in exponential phase for replication timing and gene expression studies using
standard culture conditions.Affymetrix Human Exon 1.0 STarrayswereused to
available as released from the ENCODE Project through the UCSC Genome
Browser at http://genome.ucsc.edu (see SI Methods).
Repli-Seq is based on our STS replication assay (20, 21) and has been
modified for sequence analysis of newly replicated DNA (Fig. 1). The pro-
cedure is similar for attached and unattached cell types.
The density of BrdU-DNA-derived sequence tags along the genome was
and was normalized to a global density of 4 million tags per genome for each
chromosome inactivation. Shown are two examples of biphasic replication
timing associated with X chromosome inactivation in females. In the
chrX:121.5–124 Mb region, male LCLs (H0287) have an early peak associated
with several genes known to be subject to X inactivation in females (38) (de-
pictedinred;X inactivation statusofgenes inblack isunknown).Theregionis
biphasic in female LCLs (GM06990) with the early component resembling the
male pattern (active X) and the very late component (G2) thus representing
the inactive X. In the chrX:53.1–54.7 Mb region there is a transition from a
more synchronous region containing many genes that escape X inactivation
(in green) to a biphasic region that is subject to X inactivation (in red). (B) Bi-
phasic replication in the imprinted Prader-Willi/Angelman syndrome region
on chromosome 15. Replication is markedly biphasic in hESCs over a 2–3 Mb
region associated with Prader-Willi syndrome (27), including the imprinted
SNRPN gene. The early component is specific to hESCs and correlates with the
much higher level of RNA expression in this region relative to other cell types.
(C) Biphasic patterns indicate allele-specific replication programs. Early and
late hESC fractions were examined for the presence or absence of an in-
formative restriction site polymorphism in the SNRPN gene (BstUI) to confirm
that biphasic patterns are allelic. Early-replicating DNA is exclusively the
cleavedallelethat is expressed (paternal), whereas the late-replicating DNA is
exclusively the uncleaved allele that is repressed (maternal). Controls shown
include BG02 DNA (for an equal allelic contribution) and a homozygous-
cleaved DNA control for digestion.
Biphasic replication timing. (A) Biphasic replication timing (simulta-
Hansen et al.PNAS
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| vol. 107
| no. 1
related to tag mapability variation, sequence bias, or copy-number differences, Download full-text
we further normalized the 50 kb densities of each cell line to a percentage of
total replication for that line at each genomic coordinate.
Analysis of Lineage-Specific Differences in Replication Timing and Biphasic
Replication. To simplify the computational search for variations in replication
timing in different cell lines, we combined the percent-normalized sequence
yield a cumulative “early” replication signal for each cell type. Similarly, a
“late” signal was calculated by adding the PNDV for S4 and G2. We identified
regions having significant differences in the early:late ratio between any two
cell lines and classified these as “plastic” replication domains (Table S2).
To determine regions where replication time is biphasic, cell-cycle PNDV
signals were added in pairs to generate expanded replication time categories
(G1+S1, S1+S2, S2+S3, S3+S4, S4+G2). A biphasic region was defined as a 1 kb
bin that contained at least 40% of the total replication signal in each of two
nonadjacent expanded replication time categories (Table S5).
DNaseI Sensitivity and Genomic Feature Analysis. To compare replication
timing with chromatin sensitivity to DNaseI digestion, we digested BJ
fibroblast and K562 erythroid nuclei according to previously described pro-
cedures (37), DNaseI-derived fragments were sequenced and mapped, and
150 bp densities were determined (see SI Methods for data accession).
Enrichments for particular genomic features were calculated for the dif-
interest to the genome as a whole.
ACKNOWLEDGMENTS. We thank Ping Luo (STS-based replication timing), Jeff
Goldy (cell culture), and Andrew Haydock (RNA isolation) for technical assistance.
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| www.pnas.org/cgi/doi/10.1073/pnas.0912402107Hansen et al.