What Way Does The Dna Template Strand Read
Nat Methods. Author manuscript; available in PMC 2013 May 1.
Published in terminal edited form every bit:
PMCID: PMC3580294
EMSID: EMS51847
Dna template strand sequencing of single-cells maps genomic rearrangements at high resolution
Ester Falconer
aneTerry Play a joke on Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada
Mark Hills
aneTerry Flim-flam Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada
Ulrike Naumann
1Terry Fox Laboratory, BC Cancer Bureau, Vancouver, British Columbia, Canada
Steven S S Poon
aneTerry Trick Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada
Elizabeth A Chavez
1Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada
Ashley D Sanders
1Terry Fox Laboratory, BC Cancer Agency, Vancouver, British Columbia, Canada
Yongjun Zhao
2Canada'south Michael Smith Genome Sciences Eye, BC Cancer Bureau, Vancouver, British Columbia, Canada
Martin Hirst
2Canada's Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, Canada
threeDepartment of Microbiology and Immunology, Centre for Loftier-Throughput Biology, University of British Columbia, Vancouver, British Columbia, Canada
Peter M Lansdorp
1Terry Play a trick on Laboratory, BC Cancer Bureau, Vancouver, British Columbia, Canada
fourDivision of Hematology, Department of Medicine, Academy of British Columbia, Vancouver, British Columbia, Canada
fiveEuropean Research Institute for the Biology of Ageing, University Medical Center Groningen, Groningen, The Netherlands
Abstruse
Dna rearrangements such as sis chromatid exchanges (SCEs) are sensitive indicators of genomic stress and instability, simply they are typically masked by single-cell sequencing techniques. Nosotros developed Strand-seq to independently sequence parental Dna template strands from single cells, making information technology possible to map SCEs at orders-of-magnitude greater resolution than was previously possible. On boilerplate, murine embryonic stem (mES) cells exhibit eight SCEs, which are detected at a resolution of up to 23 bp. Strikingly, Strand-seq of 62 single mES cells predicts that the mm9 mouse reference genome assembly contains at to the lowest degree 17 incorrectly oriented segments totaling nearly 1% of the genome. These misoriented contigs and fragments accept persisted through several iterations of the mouse reference genome and accept been hard to detect using conventional sequencing techniques. The ability to map SCE events at loftier resolution and fine-tune reference genomes past Strand-seq dramatically expands the scope of unmarried-cell sequencing.
Genomic instability is a major driving force of tumor development and produces copy number variations (CNVs), mutations, loss of heterozygosity and aneuploidy1. The resulting genomic heterogeneity can give proliferative and survival advantages to subsets of cells that so undergo clonal expansion2. Though existing single-jail cell deep-sequencing techniques tin can place clonal expansions by CNV signatures of individual tumor cells3, these signatures are a readout of past genomic events that accept been propagated in a significant proportion of cells in the population. Insight into the mechanisms driving tumor development will crave unmarried-cell methods that more than directly assess genome instability and genomic rearrangements.
SCEs are the effect of double-strand breaks (DSBs) repaired by homologous recombination pathways, and their accumulation is an early indicator of genomic instability4. SCEs are a diagnostic phenotype for genotoxic stresses5 and cancer-prone genetic instability syndromes such as Bloom's syndrome6. Despite the perceived importance of SCEs, it has not been possible to identify them in single cells using high-resolution sequencing approaches.
Here we report the evolution of Strand-seq, a single-cell sequencing technique that identifies the original parental Dna template strands in girl cells following prison cell sectionalisation. The method uses bromodeoxyuridine (BrdU) incorporation in the nascent strand during Dna replication followed by selective degradation of the nascent strand to isolate the template strand for structure of directional sequencing libraries.
Using Strand-seq, we identified and mapped SCEs in mES cells at a resolution orders of magnitude greater than was previously possibleseven,8. In addition, we identified aneuploidy events and CNVs in single mES cells arising from a single replication round. Notably, Strand-seq identified misoriented contigs and fragments in the current mouse reference genome assembly (mm9) that totaled nearly 25.57 Mb, or roughly i% of the genome. SCEs and contig misorientations are undetectable using conventional sequencing techniques, thus highlighting the advantage of Strand-seq in identifying and characterizing genomic instability and in fine-tuning reference genome associates. We also demonstrate that Strand-seq tin can exist used to assay single-cell template-strand inheritance on a genome-wide calibration. We anticipate that Strand-seq will be useful for haplotyping and detection of genomic rearrangements such every bit inversions and translocations that are more difficult to detect in the absenteeism of directional data.
RESULTS
Strand-seq library structure and data visualization
Strand-seq identifies parental DNA template strands in daughter cells post-obit DNA replication and cell partition. We previously designated these template strands equally Crick or Watson, corresponding to the top (forward, plus) and lesser (reverse, minus) strands, respectively, in the mouse reference genome9 (Fig. 1a). To perform Strand-seq, we cultured C2 mES cells (from an inbred C57BL/6 background) in the presence of BrdU for one round of DNA replication to create hemi-substituted genomic DNA. Nosotros then sorted single girl cells at the subsequent G1 phase of the cell cycle on the basis of the expression of a modified Fucci fluorescent cell-cycle reporter construct10 or by synchronization of the parental cells following G2 arrest11 (Supplementary Fig. 1). We fragmented the Dna by micrococcal nuclease digestion and performed custom-indexed Illumina library construction (Fig. 1a,b). Prior to PCR distension, we nicked the newly formed BrdU-substituted strands by treatment with Hoechst 33258 and UV light. The subsequent PCR amplified merely the original intact Deoxyribonucleic acid template strand, resulting in libraries in which the original genomic directionality was maintained (Fig. 1b,c). This allowed us to identify the original parental template strands from paired short sequencing reads (Fig. 1c).
Principle of single-cell Deoxyribonucleic acid template strand sequencing. (a) Top: a single parental chromosome before Deoxyribonucleic acid synthesis is shown with the Crick (blue) and Watson (orange) strands. Bottom: following DNA replication in the presence of BrdU, each sister chromatid with 1 original template strand and one complementary strand containing BrdU will segregate into i of the daughter cells. (b) Dna is fragmented and ligated to universal forked adaptors; UV photolysis creates nicks at BrdU sites, preventing PCR amplification of newly formed strands only assuasive amplification of the original intact template strand. (c) The resulting libraries are directional, containing the template strand in its original genomic orientation in all amplified fragments. Multiple single-prison cell libraries containing unique 6-nucleotide (nt) index sequences (green lines in b) are pooled and sequenced on an Illumina platform. 2 76-nt reads from both directions (red lines) will read both the original template strand (ever read i) and the complementary strand (always read 3). (d) Possible combinations of maternal (M) and paternal (P) template strands inherited by daughter cells. (east) Expected read distribution from diploid Strand-seq libraries from inbred mouse cells. Watson and Crick reads (orange and blue lines) are binned and mapped to either side of a chromosome ideogram (gray). SCEs are expected to show a switch from both Watson and Crick reads to either Watson or Crick alone (correct; arrowheads indicate SCE interval).
The nicking of BrdU-substituted DNA before PCR amplification is essential to identify parental template strands and renders Strand-seq incompatible with whole-genome distension methodsiii. Strand-seq identifies parental template strands, which can be useful for haplotyping studies. Still, the use of an inbred mouse strain precluded the identification of a parent of origin for whatsoever autosomal homolog in this written report.
We synthetic 66 indexed single-cell libraries from sorted cells (62 Strand-seq libraries and 4 standard whole-genome shotgun (WGS) libraries) that were checked for size distribution (Supplementary Fig. two) and then pooled and sequenced on an Illumina platform (Fig. 1c and Online Methods). The number of sequence reads per library subsequently quality filters were applied (come across Online Methods) ranged from threescore to 1,457 reads per Mb, which translated to genomic coverage of 0.64%–vi.46% for single-jail cell Strand-seq libraries (three.sixteen% mean) and 4.8%–eight.2% for WGS libraries (6.22% mean). The compiled genomic coverage of all 62 Strand-seq libraries was 65.56%, with ~30% of the genome covered by two or more reads. Pileups from these compiled libraries showed a periodicity consequent with nucleosomal fragments as input textile (data not shown).
Each read aligned to either the forrad or reverse direction of the reference genome, which corresponds to the original Crick and Watson strands, respectively. With the exception of the sex chromosomes, C2 mES cells from inbred mice have ii identical parental homologs of each chromosome (Fig. 1d), and reads from the template strands of both homologs from a single cell mapped to the same reference chromosome. We binned aligned reads into nonoverlapping 200-kb segments and plotted these bins equally colored horizontal lines along an ideogram of each chromosome (Fig. 1e). The length of these lines depends on the number of reads within the bin (Supplementary Fig. 3). If a girl cell inherited both Crick template strands from both parental homologues, and so simply blue lines are shown. If both Watson and Crick template strands were inherited, then both blue and orangish lines are shown (Fig. 1e). We identified SCEs resulting from mixing of template and newly formed strands during homologous recombination–based resolution of DSBs12 as points along the chromosome ideograms where reads mapping to both Watson and Crick strands switch to reads mapping to either the Watson or the Crick strand (Fig. 1e and Supplementary Fig. 4) while maintaining a consequent average read count (Supplementary Fig. iii).
High-resolution sis-chromatid-exchange mapping
Nosotros mapped paired-end sequence reads from all Strand-seq and WGS libraries (Fig. 2a; Supplementary Information contains the ideograms of all 66 private libraries). No-cell controls that underwent all steps of library construction averaged 17.one reads per Mb, indicating few contaminating reads in our single-cell libraries (Supplementary Fig. five). Inside the 62 Strand-seq libraries, we identified SCE events and mapped each exchange interval (Fig. 2b and Online Methods). Because we could not distinguish between parental homologs in this inbred mouse strain, the resolution of the commutation region was an approximation. Withal, nosotros expect it to be within an order of magnitude of our calculations because reads were distributed uniformly beyond the genome. Strand-seq of non-inbred strains or man cells volition further amend the power of SCE assay because single-nucleotide polymorphisms and haplotype mappingthirteen,14 can help identify the parent of origin of the exchanged chromatid.
Dna template strand libraries mapped to mouse chromosomes (chr) reveal SCEs. (a) Strand-seq library 3 shows Watson and Crick read distributions according to the template strands inherited from each parental homolog. SCEs (black arrowheads) are in the interval between reads that map to both Watson and Crick strands and reads which map to either strand lone. Complete switches from Watson-only to Crick-only template strand reads (red arrowheads) are potentially misoriented contigs in the reference genome (meet Fig. 3). (b) The interval betwixt Watson and Crick reads flanking the SCE can exist estimated at the base-pair level in higher-resolution screenshots from the UCSC genome browser. SCE intervals 1 and ii from a are 196 bp and 2,219 bp, respectively (see also supplementary Fig. 7). (c) All 529 SCE events in 62 Strand-seq libraries were placed into one-Mb bins and mapped to ideograms of each chromosome. (d) Frequency of SCEs per megabase is normalized for each chromosome. The average frequency across the genome for these wild-type mES cells is 0.21 SCEs per Mb (dashed crimson line). Annotation that the bodily frequency of SCEs in gray shading represents a diploid content for all autosomes and a haploid X. The SCE frequency for a diploid X (female jail cell) is extrapolated (white region). (e) Distribution of SCEs in Strand-seq libraries. All libraries contained at least three SCEs, with an average of viii SCEs per cell (dashed cherry-red line).
We binned SCEs into nonoverlapping i-Mb regions and mapped them to chromosome ideograms (Fig. 2c). SCEs were distributed along the length of each chromosome, occasionally with multiple SCE events per chromosome (Supplementary Fig. 6). A total of 517 autosomal SCE events in the 62 Strand-seq libraries were mapped to all chromosomes at a frequency of 0.21 SCE events per Mb of sequence (Fig. 2nd). Twelve chromosome Ten SCEs were also observed, which appeared as a complete switch from Watson to Crick reads every bit there is only one re-create of X in these male cells (Supplementary Fig. half dozen). The 517 autosomal SCEs were evenly distributed across the genome (Fig. 2c) with no meaning clustering or deserts at a multifariousness of bin sizes as compared to a Poisson distribution background model (P = 0.2297 for 1-Mb bin size, data not shown). On average, eight SCEs per prison cell were identified (Fig. 2e), which corresponds with counts of spontaneous SCEs in wild-type mES cells in previously published cytogenetic studies15,16. Whereas SCE mapping resolution using cytogenetic banding is on the gild of several megabases7,8, Strand-seq showed a median resolution of 5.97 kb, and one SCE consequence mapped to within 23 bp of the actual breakpoint (Supplementary Fig. seven). The loftier resolution of SCE interval mapping allows more detailed analysis of the sequences and genes surrounding the exchange interval (Supplementary Fig. eight).
Identifying misoriented regions in mm9 genome associates
Nosotros observed a striking and complete switch in template strands at exactly the same interval in chromosomes 10 and 14 (Fig. 3a and Supplementary Data) in every library in which that region inherited both Watson or both Crick template strands (a total of 24 libraries for chromosome x and 27 libraries for chromosome 14). The switch from 2 Crick to two Watson template strands cannot be explained by SCEs or translocations, as the aforementioned consequence would have had to occur on both parental homologs at the same location, in multiple cells. A monosomy combined with an SCE such every bit that observed for chromosome X (Supplementary Fig. half-dozen) could besides be ruled out because we observed typical-looking SCE events on the same chromosomes exhibiting the switches (Fig. 3b). In improver, the average read depth for chromosomes 10 and 14 in all of these libraries did not support aneuploidy (Supplementary Data). Note that these switch regions are non axiomatic if i Watson and one Crick template strand each were inherited by the daughter cell (Fig. 3c).
Strand-seq identifies contig orientation errors in the mouse reference genome. (a) A complete switch from Watson to Crick reads is observed in chromosome (chr) 10 and 14 in all Strand-seq libraries in which two Watson or Crick template strands are inherited (red arrowheads). See supplementary data for full genomic ideograms of indicated libraries. (b) Switches are not due to a monosomy of these chromosomes because typical-looking SCE switches are also observed (black arrowheads). (c) Switches are not apparent if both Watson and Crick templates are inherited. (d) The interval between the switched reads ever maps to the aforementioned unbridged gaps on chromosome ten and chromosome fourteen in the reference genome. (e) Metaphase mES cells hybridized simultaneously with 3 fluorescently labeled BAC probes xiv.1 (green), fourteen.3 (ruddy) and 14.2 (orange). Scale bar, 5 μm. (f) Signals from probes 14.ane and 14.3 should be distinct co-ordinate to the orientation of contigs flanking the gap in the reference genome; however, the fluorescence signals overlap (e, left), whereas signals from probes xiv.two and xiv.3 are distinct (east, right). (g) Corrected orientation of mm9 as inferred from Strand-seq and confirmed past FISH.
One possible explanation for these observations is that the orientation of the contigs nearest to the centromeres of chromosomes 10 and xiv was incorrectly assigned in the reference assembly. Nosotros found that in all cases, the template strand switches mapped to the aforementioned unbridged gaps between contigs in the mm9 reference genome for both chromosome 10 and 14 (Fig. 3d). Unbridged gaps are variable-sized regions of unknown sequence that are difficult to map because they contain circuitous segmental duplications and repetitive regions. Consequently, the relative orientations of contigs direct flanking these gaps take non been confirmed and are classified every bit unknown.
The mm9 genome build contains 186 unbridged gaps. To test whether Strand-seq tin can correctly predict misoriented contigs, we performed FISH17 using ii BAC probes specific for genomic regions on either end of the chromosome xiv contig and a tertiary BAC probe on the neighboring contig, which served equally a reference point (Fig. 3e–g and Supplementary Fig. 9). Probes xiv.3 and xiv.i are predicted to exist eleven.40 Mb apart in mm9, merely the probe signals overlapped in our FISH analysis, suggesting adjacency (Fig. 3e,f). Probes 14.3 and 14.2 are predicted to be 0.64 Mb apart but showed distinct fluorescence signals, indicating that they are separated past at to the lowest degree several megabases and do not directly flank the gap as in the reference genome (Fig. 3e,f). The results of the FISH analysis of chromosome x are similar, thus supporting our hypothesis of contig orientation errors (Supplementary Fig. ix).
To ostend that these findings are not genomic rearrangements unique to the C2 groundwork, we repeated FISH analysis in 3T3 murine fibroblasts with a Swiss albino genetic background and obtained identical results (Supplementary Fig. 9). These findings suggest that the orientation of the contigs {"blazon":"entrez-nucleotide","attrs":{"text":"NT_039490.vii","term_id":"149260621","term_text":"NT_039490.7"}}NT_039490.vii on chromosome ten and {"blazon":"entrez-nucleotide","attrs":{"text":"NT_039595.7","term_id":"149265172","term_text":"NT_039595.seven"}}NT_039595.7 on chromosome 14 in mm9 should be reversed (Fig. 3g). We also observed smaller regions of complete template strand switches (Supplementary Fig. 10). In total, 17 contig fragments totaling nearly i% of the genome are predicted to be incorrectly oriented according to Strand-seq (Table 1), ranging in size from 166.8 kb to thirteen.1 Mb (Supplementary Table ane). Most of these fragments are much smaller than the 2-Mb resolution limit of FISH.
Tabular array ane
Misoriented genomic regions of mm9 genome assembly
| Classification | No. of fragments | Size (bp) | Proportion of genome (%) |
|---|---|---|---|
| Misoriented | 17 | 25,482,119 | 0.97 |
| Unknown orientation | 18 | 6,192,462 | 0.22 |
| Correctly oriented | 148 | 2,654,895,218 | 98.81 |
Orientations of genomic regions of mm9 genome assembly as classified by Strand-seq.
Comparison to previous releases of the mouse reference genome showed that some predicted fragment misorientations were corrected in subsequent assemblies, whereas others remain unresolved (Supplementary Fig. 11). We observed these misoriented fragments in every library with a Watson-just or Crick-only template-strand inheritance pattern in these regions, with no discrepancies (Supplementary Table 1a). We were unable to determine the orientation of xviii unbridged fragments (totaling 0.22% of the genome) because of poor coverage or complex segmental duplications that prevented strand-specific alignment of short sequencing reads in those regions (Supplementary Table 1b). This analysis confirms that the remaining 148 genomic fragments that flank unbridged gaps are correctly oriented in the reference genome, effectively 'bridging' these gaps. Of note, Strand-seq libraries reveal SCEs and misoriented fragments, whereas WGS libraries mask such features (Supplementary Fig. 12); Strand-seq is therefore a valuable tool for fine-tuning reference genome assemblies.
We were also able to detect genomic duplications and aneuploidy in both our Strand-seq and WGS libraries without PCR amplification of input textile (Supplementary Fig. 13). The aggregating of aneuploidy is a well-known miracle in continually cultured mES cells18, and 17 of our 66 full libraries displayed at to the lowest degree 1 aneuploidy upshot (Supplementary Data). For example, one cell (library iv) showed a duplicated region in chromosome iv as well equally trisomy of chromosome 5 and monosomy for chromosome 10. These duplication and aneuploidy events were evident in both the Strand-seq and WGS library constructed from the same single jail cell (Supplementary Fig. 13), indicating that our libraries tin appraise genomic CNVs in single cells19 without the bias that could be introduced by PCR amplification of genomic DNA20.
Give-and-take
Unmarried-cell DNA template strand sequencing (Strand-seq) provides high-resolution maps of SCEs, identifies other indicators of genomic instability such as aneuploidy and CNVs, and identifies misoriented fragments in the mouse reference genome assembly. The contribution of SCEs to tumor heterogeneity is considered secondary to that of other chromosomal abnormalities such as translocations and CNVs, likely because SCEs are thought to be error-free recombination events ensuing from replication-fork plummet. However, unequal crossing over in SCEs can pb to CNVs, loss of heterozygosity and aneuploidy1. Importantly, a high number of SCEs is an indicator of aggregating of DSBs during replication, a symptom of replication stress due to complanate replication forks, or the inability of the Dna repair pathways to suppress homologous recombination to repair DSBs (as in Bloom's syndrome)5. Therefore, SCE mapping at high resolution will be a valuable contribution to the analysis of tumor evolution and the progression of genomic instability in replicating cells.
Although we cannot exclude the contribution of BrdU to the formation of DSBs or to the resolution of SCEs in our arroyo (nor in traditional cytogenetic assays of SCEs requiring two rounds of BrdU incorporation)5, Strand-seq can exist used to finely map spontaneous SCEs in cells that undergo replication stress from genotoxic or chemotherapeutic agents, radiation, mutations in Dna repair and recombination pathways, or other genomic instability events. Unlike cytogenetic techniques, Strand-seq can provide in-depth assay of frail sites or other characteristics of genomic sequences surrounding breakpoint regions. In addition, the method requires only one mitotic cycle in the presence of BrdU, which is ideal for studies of SCE in vivo.
Nosotros accept demonstrated that Strand-seq tin can exist used to orient unbridged contigs that tin occur in regions that are difficult to assemble, such as circuitous segmental duplications and repetitive regions. This written report provides contig orientation data for 99.78% of the genome associates from a relatively small-scale data ready (Supplementary Fig. 10c). The importance of correctly oriented contigs is highlighted by disease clan studies that rely on the correct location of markers to identify candidate genes—the results of which could be complicated by regions that are misoriented. In our study, the misoriented contig on chromosome fourteen is large enough to show a discrepancy between physical and genetic map distance, which has been erroneously attributed to a break-down in linkage disequilibrium due to meiotic recombination21. It will exist important to confirm the orientation of fragments in other genomes, including those flanking the 271 unbridged gaps present in the homo genome.
Strand-seq is the ideal technique to report template strand inheritance in social club to exam nonrandom segregation of sister chromatids, as was proposed for chromosome 7 in mES cells22. Notwithstanding, the prevalence of SCEs as well as aneuploidy events in all the unmarried cells that we sequenced prevented the consignment of Watson or Crick template strands for many chromosomes (Supplementary Fig. 14). Nevertheless, if we exclude these chromosomes from analysis, we detect no deviation from a random segregation pattern for chromosome 7 in mES cells equally judged by χ 2 analysis (Supplementary Table two and information not shown). The occurrence of SCEs also suggests that it is not valid to employ minor probes to correspond the template strands of entire chromosomes (as in recent template-strand segregation studies9,23) because the mixing of template and nontemplate strands in SCEs is ignored (Supplementary Fig. 14c). Furthermore, unless stem cells are demonstrated to completely suppress SCEs, it is not possible to claim completely asymmetric template-strand segregation to support, for example, the immortal strand hypothesis24,25.
Other expected applications of Strand-seq are the phasing of alleles to establish parental haplotypesthirteen,fourteen and the mapping of inversions, translocations and other chromosomal abnormalities26,27 in unmarried cells without using the big amounts of input material or the depth of sequencing currently required in existing sequencing approaches28,29. When one Watson and one Crick template strand is inherited from each parent, those strands are already phased considering they originate from different parental chromosomes. We await that Strand-seq volition serve as a powerful tool to study genetic rearrangements in single cells during development, cancer and aging.
ONLINE METHODS
Jail cell culture
Undifferentiated wild-type murine embryonic stem cells (C2, C57BL/6 background) were cultured every bit described9. Murine embryonic fibroblasts were grown in DMEM-FCS. For training of metaphase cells, colcemid (Sigma-Aldrich, 0.1 μg/ml) was added one h before harvest. Trypsinized cells were treated with 0.075 Chiliad KCl for 10 min before fixation with iii:1 methanol/acerb acrid using standard cytogenetic procedures. Fixed cells were stored at −20 °C.
A modified Fucci reporter construct was cloned by linking the jail cell-cycle reporters from the pFucci-G1 Orangish and pFucci-Southward/G2/M expression vectors (MBL International) with a self-cleaving T2A peptidexxx. The Fucci construct was transfected into C2 cells using Effectene Reagent (Qiagen), and cells were selected using puromycin and repeated FACS sorting. Cycling between cell-cycle colors was confirmed by acquisition of fourth dimension-lapse movies on a Coolsnap HQ digital camera attached to an inverted microscope (IX70 Olympus) fitted to a DeltaVision RT imaging system (Practical Precision) equipped with appropriate filter sets. Movies confirm ES-cell accumulation of magazine during the S, G2 and M stages of the cell bicycle, punctuated by cytokinesis and followed by mKO fluorescence in the G1 girl cells (information non shown). BrdU (Invitrogen) was added to semiconfluent cultures at a final concentration of 40 μM for 8–12 h earlier harvest.
G2 synchronization of mES cells
C2 ES cells alone or with the Fucci reporter construct were synchronized at the G2 phase by treatment with x μM (terminal) RO-33066 (ref. eleven) for 4 h, which was followed by release into twoscore μM (final) BrdU for 16 h.
FACS sorting and genomic Deoxyribonucleic acid fragmentation
To analyze DNA content, 10 μg/ml Hoechst 33342 (Sigma-Aldrich) was added to the cell civilisation thirty min before harvest. The dye was besides nowadays in the FACS buffer. Cells were trypsinized, resuspended in phosphate-buffered saline with 2% FCS and sorted on a BD Influx cell sorter (BD Cytopeia) equipped with two tunable Coherent I305C argon lasers and a Cobolt Jive 50 561-nm diode laser.
Single cells were sorted directly into 100 μl lysis buffer (nuclei isolation buffer, NucleiEZ kit, Sigma) in flexible unskirted PCR plates (Bio-Rad) fitted into a rigid plate holder for sorting and spinning. Plates were immediately spun in a four °C prechilled centrifuge at 500k for five min to pellet nuclei. Plates were carefully removed from adaptors, and 90 μl cell-lysate supernatant was removed slowly and carefully using a long flexible gel-loading tip in order to avoid aspirating the nucleus. Next, xl μl of i.25× micrococcal nuclease (MNase) primary mix (62.5 mM Tris-HCl pH seven.9, 6.25 mM CaCl, 0.03125 U/μl MNase enzyme, New England Biolabs) was added to each well containing a nucleus (as well equally to no-prison cell negative-command wells containing only lysis buffer). Reactions were mixed 20–30 times using a pipettor and incubated at room temperature for 5 min. Reactions were stopped by calculation 5.5 μl 100 mM EDTA (10 mM final) and mixing xx–thirty times with a pipettor. The digested chromatin was transferred from the PCR plate into clean microcentrifuge tubes. Each well was rinsed with 100 μl buffer EB (Qiagen) and added to each tube. DNA was extracted by calculation an equivalent amount (155 μl) of 25:24:1 ultrapure phenol:chloroform:isoamyl booze (Invitrogen) to each tube, mixing well and spinning at 13,000 r.p.yard. for v min at room temperature in a benchtop microcentrifuge. And then 150 μl of the top aqueous layer containing extracted Dna fragments was removed to a make clean microcentrifuge tube and precipitated with 0.1 vol. 3 M sodium acetate solution (Sigma-Aldrich) and ii.v vol. 100% ethanol (EMD) with 1.5 μl linear polyacrylamide (GeneElute LPA, Sigma-Aldrich) added as a coprecipitant. Tubes were incubated at −20 °C for 20 min and centrifuged at fourteen,000 r.p.k. for 30 min. at 4 °C. Supernatant was advisedly removed, and the pellet was washed once with 70% ethanol and then dried at room temperature. DNA was reconstituted in 20 μl EB for library construction.
Dna template strand library construction
Library construction for the Illumina sequencing platform was performed using a modified paired-end protocol (Illumina). This involved end-repair and A-tailing of fragmented Deoxyribonucleic acid followed by ligation to Illumina PE adaptors and PCR amplification. At each step in the procedure, reactions were purified using either phenol:chloroform:isoamyl alcohol extraction followed by ethanol precipitation or solid-phase reversible immobilization paramagnetic beads (Agencourt AMPure, Beckman Coulter). 1 μM of Illumina PE adaptors were ligated to A-tailed Deoxyribonucleic acid fragments at a terminal concentration of 33.five nM for fifteen min at room temperature using 5,000 units of Quick T4 ligase (New England Biolabs). Ligation products were purified using 0.viii vol. Agencourt AmpureXP magnetic chaplet (Beckman-Coulter) and eluted in 11 μl or 22 μl EB buffer (Qiagen). To create nicks in the BrdU substituted Deoxyribonucleic acid strands, eluted Dna was incubated with 10 ng/μl Hoechst 33258 (Sigma-Aldrich) for 15 min at room temperature in clear 0.25-ml PCR tubes (Rose Scientific) protected from calorie-free. PCR tubes were then uncapped, and DNA was treated with UV for 15 min (the calculated dose was 2.7 × ten3 J/1000ii). Nicked DNA was then used as a template for PCR using Phusion HF primary mix (NEB) and primers PE ane.0 (Illumina) and a custom multiplexing PCR primer five′-CAAGCAGAAGACGGCATACGAGATNNNNNNNCGGT CTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3′, where 'NNNNNN' was replaced with unique fault-tolerant hexamer barcodes. The PCR program was every bit follows: initial denaturation of 98 °C, thirty s; xv cycles of (98 °C, 10 s; 65 °C, 30 southward; 72 °C, 30 s); and terminal extension of 72 °C, 5 min. PCR products were purified using 0.8 vol. AmpureXP beads and eluted in 11 μl EB. one μl library was run on an Agilent High Sensitivity chip (Agilent) to check size distribution before pooling for sequencing.
Illumina sequencing
Libraries were pooled for sequencing, and the 200- to 400-bp size range was purified away from adaptor ligation artifacts on an 8% Novex TBE Page gel (Invitrogen). Deoxyribonucleic acid quality was assessed and quantified using an Agilent DNA 1000 serial II analysis (Agilent) and Nanodrop 7500 spectrophotometer (Nanodrop) and afterward diluted to ten nM. The terminal concentration was confirmed using a Quant-iT dsDNA HS assay kit and Qubit fluorometer (Invitrogen). For sequencing, clusters were generated on the Illumina cluster station (GAIIx) or cBOT (Hiseq2000), and paired-end 76-nt reads were generated using v4 sequencing reagents on the Illumina GAIIx (v4) or Hiseq2000 (SBSxx) platform following the manufacturer's instructions. Between the paired 76-nt reads, a third 7-bp read was performed using the custom sequencing primer five′-GATCGGAAGAGCGG TTCAGCAGGAATGCCGAGACCG-3′ to sequence the hexamer barcode. Image assay, base of operations-calling and error scale were performed using Illumina's genome-analysis pipeline.
Bioinformatic analysis
Indexed paired-end .qseq files were aligned to the mouse reference genome (mm9) using bwa31, and custom scripts were used to split the resulting .bam files by alphabetize and to add the guiltlessness flag. The resulting .bam files were sorted and filtered for duplicates (which removes both single-end and dual-finish duplicates) and low-quality alignments (q < 20) using Samtools Version 0.1.10 (ref. 32). We developed a pipeline, Bait (bioinformatic analysis of inherited templates), that parsed the bam files on the ground of the strand directionality assigned to each read. Reads that mapped to the '+' strand from the first PET (paired-cease tag) and the '−' strand reads from the second PET were classified as Watson reads, and reads that mapped to the – strand from the first PET and the + strand from the second PET were classified every bit Crick reads. These data were plotted as separate histograms against ideograms of mouse chromosomes, with reads counted in 200-kb bins across each chromosome. Boosted files in .bed format were plotted over the ideograms to represent sequence gaps and contig orientations. The number of reads mapping to Watson or Crick for each chromosome were summed, and the number of reads per megabase for each chromosome was calculated and printed below the ideograms. Normalized counts per megabase were determined past calculating the sum of both Watson and Crick reads for all autosomes and dividing by the length of the autosomes (in megabases). Any chromosomes in which read counts were 0.66× lower or 1.33× college than the normalized count were classified as monosomies or trisomies, respectively. SCE events were defined as the interval in which there was a switch from reads mapping to both Watson and Crick strands to reads mapping to merely ane of the strands, without a corresponding change in the total number of reads such that the sum of Watson and Crick reads remained constant. Our criteria further stipulated that there must be ten consecutive Watson-simply or Crick-only reads subsequently the interval switch to count the switch as an SCE or to confirm fragment or contig orientation. To verify SCE and misorientation events, the SCE and misoriented contig interval coordinates were also converted to .bed files using BEDtools33 and uploaded to the UCSC genome browser to identify genomic features and genome build features, such as contigs, and to make up one's mind suitable BACs for FISH probes.
Fluorescence in situ hybridization analysis
Metaphase chromosomes from C2 ES cells and prematurely condensed chromosomes34 from murine 3T6 fibroblast cells were prepared and used for three-colour FISH. BAC probes from chr x or chr 14 were labeled using a nick translation kit (Abbott Molecular) with Spectrum-Green dUTP (probe 10.1: RP23-38N9 and probe14.1: RP23-452I3), Spectrum-Orange dUTP (probe x.2: RP23-128M21 and probe 14.2: RP23-154F13) and Cherry-red dUTP (probe 10.three: RP24-258P4 and probe14.three: RP23-255D5) according to manufacturer instructions. Hybridization and image analysis were performed as described previously17.
Fluorescence microscopy, prototype conquering and option
Fluorescence signals were captured on an Axioplan microscope (Zeiss) equipped with filters for DAPI, FITC, Cy3, Cy5 and Texas Crimson (Chroma Engineering science and Semrock) using an Axiocam MRm digital camera controlled by Metasystems ISIS software (Altlussheim). Alternatively, images were caused on a Coolsnap HQ digital camera attached to an inverted microscope (IX70 Olympus) fitted to an imaging system (DeltaVision RT, Applied Precision) equipped with similar filter sets. Grayscale (12-bit) images at the wavelengths of involvement were caused through a high–numerical aperture 63×/1.4-N.A. or 60×/1.4-N.A. oil-immersion lens.
Supplementary Cloth
SI
ACKNOWLEDGMENTS
We thank J. Brind'Amour and S. Rentas for discussions and J. Schein and C. Carter (Genome Sciences Centre) for BACs. We also give thanks K. Gan for help with preliminary MNase experiments. U.Due north. was supported by a Fellowship for Prospective Researchers from the Swiss National Scientific discipline Foundation (project no. PBBEP3_131554). Work in the Hirst laboratory is supported by Canadian Institutes of Health Research grant RMF-92093. Piece of work in the Lansdorp laboratory is supported by grants from the Canadian Institutes of Health Research (RMF-92093 and 105265), the United states National Institutes of Health (R01GM094146) and the Terry Fox Foundation (018006). P.One thousand.Fifty. is a recipient of an Advanced Grant from the European Research Council.
Footnotes
Accession codes. Sequencing information accept been deposited in the Sequence Read Archive: SRA055924.
Note: Supplementary information is available in the online version of the paper.
Methods and whatsoever associated references are bachelor in the online version of the paper
COMPETING FINANCIAL INTERESTS
The authors declare no competing financial interests.
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Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3580294/
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