Stranded short nascent strand sequencing reveals the topology of DNA replication origins in Trypanosoma brucei
- University of Montpellier, CNRS, IRD, Academic Hospital (CHU) of Montpellier, MiVEGEC, France
- Trypanosoma unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Belgium
- MGX-Montpellier GenomiX, University of Montpellier, CNRS, INSERM, France
Figures
Figure 1
Experimental protocol and bioinformatics workflow for selecting the origins.
(A) Schematic representation of the experimental workflow for short nascent strand (SNS) purification. A replication bubble with DNA replication intermediates and their orientation is shown at the top. The methodology employed for the generation of the SNS-enriched samples and corresponding SNS-depleted controls is illustrated. (B) Workflow of stranded library preparation for Illumina sequencing. Single-stranded DNA fragments were tailed and tagged at the 3' end with truncated adaptor 1. Primer extension was used to generate the complementary strand. The ligation step was used to add the truncated adaptor 2. The indexing PCR step included full-length adaptors. (C) Schematic diagram of the bioinformatic workflow applied for stranded SNS-seq data analysis. Two steps of peak selection were performed to map the origins. The first step was peak calling against SNS depleted control. The second step was peak filtering, based on the three indicated criteria. (D) The schematic representation shows one origin, generating a replication bubble with two SNS leading strands in divergent orientation (SNS +and SNS –). The plus and minus SNSs are presented to illustrate the growth of the leading strand in both directions after the ligation of the upstream Okazaki fragments to the 5’ ends of SNS. The different sizes of the isolated SNS are also presented. The pink peak represents the SNS minus peak synthesised on the plus DNA strand. The blue peak represents the SNS plus peak synthesised on the minus DNA strand. The dashed rectangle, situated between the two inner borders of two divergent SNS peaks, and the green box represent the mapped origin.
Figure 2 with 2 supplements
Genomic distribution of mapped origins.
(A) A representative screenshot from Integrative Genomics Viewer (IGV) presenting the read coverage and the positions of the mapped origins in a~80 kb long genomic region of the core chromosome 1. The three replicates of BSF cells are presented. The total read coverage is in black, the minus and plus strand read coverage are in pink and blue, respectively. The green bars represent the mapped origins. The blue bars show the position of the genes and the direction of transcription of the polycistronic unit is indicated by the blue arrow below. (B) The pie charts show the proportions of mapped origins within the core (yellow) and non-core (green) regions in PCF and BSF cells, as well as the distribution of these two regions within the genomic sequence. (C) Heatmaps present the distribution of the mapped origins and shuffled controls within centred genic and intergenic regions (±5 kb). Shuffled controls present random genomic regions chosen with respect to the size and chromosomal distribution of origins. (D) The pie charts illustrate the proportions of the mapped origins of PCF and BSF cells within four indicated genomic regions, as well as the distribution of these four regions within the genomic sequence. The p-values were computed using Chi-square tests, which involved a comparison of the absolute numbers of indicated categories for each pair of datasets (**** - p<0.0001). (D) Empirical cumulative distribution function (ECDF) showing the proportion of pairwise distances between origins (pink) or between shuffled control regions (green). The p-values were computed using Chi-square tests.
Figure 2—figure supplement 1
Quantification of read count, called and filtered peaks and detected origins.
(A) Number of reads obtained by paired-end sequencing (starting number), after trimming and duplicate removal and after mapping of reads against T. brucei 427–2018 genome. (B) Number of peaks after peak calling step (bars in blue) and after peak filtering step (bars in orange). Three replicates of PCF and BSF cells are presented. (C) Proportion of the filtered peaks compared to the called peaks (100%). Three replicates of PCF and BSF cells are presented. (D) Overlap of mapped origins between three replicates of PCF and BSF cells (±100 bp window). The number of mapped origins in three replicates of PCF and BSF cells and their overlap are shown above the bars. The overlap is represented by grey bars.
Figure 2—figure supplement 2
Quantitative Features of Replication Origins.
(A) Violin-plots present the distribution of the lengths of mapped origins for the three replicates of PCF and BSF cells. The white line represents the median value. The dotted lines represent the 25th (Q1) and 75th (Q3) percentiles of the measurements. (B) The graphs present the proportions of clustered and single origins within genomic regions of indicated lengths for PCF (left) and BSF (right) cell lines. (C) Venn diagram showing the number of mapped origins in PCF and BSF cells, with the number of common origins for both cell types. Direct overlap was calculated. (D) Bar plots of the normalised numbers of detected origins in PCF (green) compared to BSF (pink). For the normalisation, all reads of the three replicates of BSF and PCF were combined respectively and the number of reads in PCF was down-sampled 15 times so that the number of mapped reads match the one in BSF. Afterwards strand-specific peak-calling against normalised SNS-depleted control was performed, followed by peak filtering and origin detection, as described in the Methods and in the main text. The mean for the 15 down sampled PCF subsamples was calculated; the error bars represent the standard deviation. The p-value was calculated using a t-test (**** - p<0.0001). (E) Distribution of population based inter-origin distances of PCF and BSF cells represented as violin plots, green for PCF cells and pink for BSF cells. The white line represents the median value. The dotted lines present the 25th (Q1) and 75th (Q3) percentiles of measurements. A two-tailed Mann-Whitney test was used to compute the p-value (* - p<0.05). The table shows the minimum (Min.) and maximum (Max.) value, the 25th (1st Qu.) and 75th (3rd Qu.) percentiles as well as the median and mean values. The measurements are indicated in base pairs.
Figure 3
Comparison of DNA replication parameters between PCF and BSF cells by DNA molecular combing.
(A) The figure depicts representative DNA molecules after immuno-detection. The immuno-detected DNA is indicated in blue, the initial pulse of nucleosides (IdU) in red, and the subsequent pulse of nucleosides (CldU) in green. The lower panels display only the red and green tracks from the first and second pulse of nucleosides extracted from the corresponding upper panels. 50 kb scale bars are shown as white lines. (B) The velocity of the replication forks was calculated by dividing the length of the CldU tracks with the duration of the CldU pulse on intact DNA molecules (schema on upper panel). Violin plots present distribution of the measured replication fork velocities in two cell types. (C) The inter-origin distance (IOD) was defined as the length between two adjacent replication initiation sites and can be determined by measuring the centre-to-centre distances between two adjacent progressing forks (schema on upper panel). Violin plots present the distribution of the measured IODs in two cell types. (D) The upper panel presents the concept of long fork/short fork ratios. The long fork/short fork ratio corresponds to the ratio of the longer IdU + CldU signal length over the shorter IdU + CldU signal length of bidirectional replication forks. A ratio >1 indicates fork asymmetry, while a ratio = 1 indicates fork symmetry (Stanojcic et al., 2016). The lower panel presents the distribution of the measured asymmetry of replication forks in two cell types. (E) The upper panel presents the concept of the measurements of the analysed DNA lengths. The lower panel presents violin plots with the measured lengths of the analysed DNA in two cell types. White bars on the violin plots indicate median values. Dotted black lines indicate quartiles. Two-tailed Mann-Whitney test was used to compute the corresponding p-values (ns - non-significant; * - p<0.05; ** - p<0.01; *** - p<0.001; **** - p<0.0001). The number of measurements (Nb) is given for each cell type.
Figure 4 with 3 supplements
Spatial organisation of nucleotides and polynucleotides in the vicinity of origins.
(A) The profile plots illustrate the distribution of four nucleotides around centred origins and shuffled controls (±2 kb). The nucleotide percentage was calculated within 20 bp window. (B) The profile plots illustrate the distribution of four and eight polynucleotides around centred origins and shuffled controls (±2 kb). A smoothing function was employed to calculate the mean frequency of polynucleotides per position within a 100 bp window (50 bp up- and downstream from the position). (C) The proportions of origins and shuffled controls without or with four or eight As upstream and/or four or eight Ts downstream of the centre. The ±0.5 kb window from the centre was analysed. The p-values were calculated using the Chi-square test, which involved a comparison of the absolute numbers of indicated categories for each pair of datasets (**** - p<0.0001).
Figure 4—figure supplement 1
Nucleotide and Polynucleotide distribution in the vincinity of origins.
(A) Profile plots and heatmaps presenting the distribution of the four nucleotides around centred origins (blue line in the profile plot) and shuffled controls (green line in the profile plot). A region of ±2 kb from the centre was analysed. The nucleotide percentage was calculated within 20 bp windows. (B) A graphical representation of nucleotide occurrence. Sequence logo of the centred origins (±250 nt) generated with WebLogo 3 47 (https://weblogo.threeplusone.com/). The arrow indicates the centre of the averaged origins. (C) Profile plots showing polynucleotide frequency around centred origin (left panels) and shuffled controls (right panels). A region of ±2 kb from the centre was analysed. The frequency of four to eight polynucleotides (4 N-8N) is shown as indicated.
Figure 4—figure supplement 2
Nucleotide and Polynucleotide distribution in the vincinity of PCF and BSF origins.
(A) The profile plots illustrate the distribution of four nucleotides around centred origins from PCF and BSF cells separately and corresponding shuffled controls (±2 kb). The nucleotide percentage was calculated within 20 bp windows. The mapped origins in the PCF and BSF cells were analysed separately to show that they can be compared with the merged (PCF +BSF) set of origins shown in the main figures. (B) Profile plots showing polynucleotide frequency around centred origin from PCF and BSF cells (left panels) and corresponding shuffled controls (right panels). A region of ±2 kb from the centre was analysed. The frequency of four and eight polynucleotides (4 N and 8 N) is shown as indicated. The mapped origins in the PCF and BSF cells were analysed separately to show that they can be compared with the merged (PCF +BSF) set of origins shown in the main figures.
Figure 4—figure supplement 3
The profile plots show nucleotide distributions around two sets of regions derived from the same stranded SNS-seq experiments.
(A) The merged origins (PCF +BSF), mapped with stranded SNS-seq approach in the middle of two divergent SNS peaks (see Methods), and their corresponding shuffled controls (±2 kb) are shown. (B) Total called peaks, which would represent origins in a conventional (non-stranded) SNS-seq approach, along with their respective shuffled controls (±2 kb) are shown. The nucleotide percentages were calculated in 20 bp windows.
Figure 5 with 1 supplement
Distribution of G4 structures in the vicinity of origins.
(A) The profile plots and heat maps show the distribution of the experimentally obtained G4 structures around centred origins and intergenic shuffled controls (±2 kb) in the Tbb TREU927 reference genome (Methods). The plus strand (light blue) and minus strand (pink) G4s, obtained under physiological conditions and in the PDS drug stabilised condition (Marsico et al., 2019) were overlapped with origins mapped by stranded SNS-seq. Mean G4 score presents average G4 score (Marsico et al., 2019) per 20 bp window. (B) The proportions of origins that lack or possess G4 structures on one or both sides of the centre were determined. The ±2 kb window from the centre was analysed for the presence of G4s. Two sets of experimental G4 structures (Marsico et al., 2019) were subjected to analysis in comparison with intergenic shuffled controls. The p-values were calculated using the Chi-square test, which involved a comparison of the absolute numbers of indicated categories for each pair of datasets (**** - p<0.0001). (C) Empirical Cumulative Distribution Function (ECDF) of the distances between the origins and the closest physiological and stabilised G4 structures (Marsico et al., 2019). Median distances are indicated. (D) The profile plots illustrate the distribution of the poly(dA) sequence (AAAA; dashed lines) and the experimental G4s (physiological and PDS drug stabilised; Marsico et al., 2019) (solid lines) around centred origins and intergenic shuffled controls (±2 kb). The analysis was conducted in the Tbb TREU927 reference genome (Methods). The plus strand is represented in blue and the minus strand in pink. A smoothing function was employed to calculate the accumulated counts of the AAAA and G4s per position within a 100 bp window (50 bp up- and downstream from the position).
Figure 5—figure supplement 1
Distribution of G4 structures in the vincinity of PCF and BSF origins.
(A) The profile plots and heat maps show the distribution of the experimentally obtained G4 structures around centred origins and intergenic shuffled controls (±2 kb) in the Tbb TREU927 reference genome. The mapped origins in the PCF and BSF cells were analysed separately to show that they can be compared with the merged (PCF +BSF) set of origins shown in the main figures. The plus strand (light blue) and minus strand G4s (pink), obtained under physiological conditions and in the PDS drug stabilised condition (Marsico et al., 2019) were overlapped with the stranded SNS-seq mapped origins. Mean G4 score presents average G4 value (Marsico et al., 2019) per 20 bp window. (B) The proportions of origins (merged PCF +BSF) and corresponding shuffled control regions that lack or possess G4 structures on one or both sides of the centre are shown in percentage. A±0.5 kb window from the centre was analysed. Two sets of experimental G4 structures (physiologic G4s and stabilised G4s; Valton and Prioleau, 2016) were analysed in origins and intergenic shuffled control. The p-values were calculated using the Chi-square test, which involved a comparison of the absolute numbers of three indicated categories for each pair of datasets (**** - p<0.0001). (C) The proportions of origins that lack or possess G4 structures (Marsico et al., 2019) on one or both sides of the centre were determined. The mapped origins in the PCF and BSF cells were analysed separately to show that they can be compared with the merged (PCF +BSF) set of origins shown in the main figures. The ±2 kb region from the centre was analysed for the presence of G4s. Two sets of experimental G4 structures, physiological and stabilised (Marsico et al., 2019) were subjected to analysis in comparison with an intergenic shuffled control. The p-values were calculated using the Chi-square test, which involved a comparison of the absolute numbers of indicated categories for each pair of datasets (**** - p<0.0001).
Figure 6 with 1 supplement
The distribution of nucleosomes around the origins in T. brucei.
(A) The profile plot and heatmaps show the distribution of nucleosomes (MNase-seq data) detected in PCF and BSF cells (Maree et al., 2017) around the centred origins (blue line) and the intergenic shuffled controls (green line; ±2 kb) in Tb Lister 427 reference genome. One replicate of MNase-seq data (Maree et al., 2017) for PCF (GSM2407366) and one replicate for BSF cells (GSM2407365) is shown here, the other replicates are presented in Figure 6—figure supplement 1A. Mean nucleosome occupancy presents average nucleosome score (dyad value; Maree et al., 2017) per 20 bp window. (B) The percentage of origins and intergenic shuffled controls with or without high nucleosome occupancy (HNO) and low nucleosome occupancy (LNO) regions in the indicated combinations. Quantification was performed for the same replicates as indicated in the Figure 6A. The p-values were calculated using the Chi-square test, which involved a comparison of the absolute numbers of indicated categories for each pair of datasets (**** - p<0.0001). (C) Percentage of the three specified categories of HNO and LNO combinations that were significantly different between origins and shuffled controls. The remaining three categories were not statistically significant. The numbers indicate mean percentage values. The bars indicate standard deviations. Mann Whitney two-tailed test was performed to calculate p-values (*** - p<0.001). (D) The profile plots show the distribution of the poly(dA) sequence (AAAA; dashed line), the predicted G4 structures (solid line) and the nucleosome distribution of the replicate GSM2407365 from BSF cells (Maree et al., 2017; blue dotted line) around centred origins and intergenic shuffled controls (±2 kb). The G4s were predicted by the G4Hunter application (Brázda et al., 2019) in the Tb Lister 427 reference genome. The plus strand is represented in green, and the minus strand in pink. The values on the plots present the accumulated counts of the AAAA, G4s and nucleosome occupancy scores per position within a 100 bp window (50 bp up- and downstream from the position).
Figure 6—figure supplement 1
Distribution of nucleosomes around PCF and BSF origins.
(A) Profile plots and heatmaps presenting the distribution of nucleosomes around the centred origins and shuffled controls. The region of ±2 kb from the centre was analysed. Eight replicates of MNase-seq data (four PCF replicates [GSM2407366, GSM2407367, GSM2407370, and GSM2407371] and four BSF replicates [GSM2407364, GSM2407365, GSM2407368, and GSM2407369]) describing the distribution of nucleosomes (Maree et al., 2017) were analysed. The upper profile plots depict the origins and shuffled controls, which were represented in blue and green, respectively. Mean nucleosome occupancy presents average nucleosome score (dyad values; Maree et al., 2017) per 20 bp window. (B) The proportions of origins and shuffled controls with or without high nucleosome occupancy (HNO) region on one or both sides of the centre and a low nucleosome occupancy (LNO) at the centre of the regions in different combinations are shown in stacked bar plots. The proportions were calculated for the eight replicates of nucleosome positioning as indicated (Maree et al., 2017). The p-values were calculated using the Chi-square test, which involved a comparison of the absolute numbers of six indicated categories for each pair of datasets (**** - p<0.0001).
Figure 7 with 1 supplement
The distribution of DRIP-seq enrichment, splice acceptor sites (SAS), and polyadenylation sites (PAS) around the origins.
(A) The profile plots and heatmaps show the distribution of R-loops (Briggs et al., 2018) around the centred origins (blue line) and the intergenic shuffled controls (green line; ±2 kb). Mean DRIP-seq signal presents average DRIP-seq signal (Briggs et al., 2018) per 20 bp window. (B) The proportion of origins and shuffled controls that overlap with R-loops (intersection without window). The p-values were calculated using Fisher’s exact (two-sided) test, which involved a comparison of the numbers of indicated categories for each pair of datasets (**** - p<0.0001). (C) The profile plots illustrate the distribution of the stranded poly(dA) sequence (AAAA; dashed line), predicted G4s (solid line) and R-loops (Briggs et al., 2018; solid blue line) around centred origins and shuffled controls (±2 kb). The plus strand is represented in green, and the minus strand in pink. The 13,409 G4s were predicted by the G4Hunter application (Brázda et al., 2019) in the Tb Lister 427–2018 reference genome. The values on the plots present the accumulated counts of the AAAA, G4s and R-loop signals per position within a 100 bp window (50 bp up- and downstream from the position). (D) Left panel: the profile plots and heatmaps show the distribution of transcription splice acceptor sites (SAS) around the centred origins (blue line) and the intergenic shuffled controls (green line; ±2 kb). Intersection was performed in the Tbb TREU927 reference genome for the 11 megabase chromosomes. Right panel: the proportion of origins and shuffled controls that overlap with SAS (intersection without window). The p-value was calculated using Fisher’s exact (two-sided) test, which involved a comparison of the numbers of indicated categories for each pair of datasets (**** - p<0.0001). (E) Left panel: the profile plots and heatmaps show the distribution of transcription polyadenylation sites (PAS) around the centred origins (blue line) and the intergenic shuffled controls (green line; ±2 kb). Intersection was performed as in Figure 7D. Right panel: the proportion of origins and shuffled controls that overlap with PAS. The p-values were calculated using Fisher’s exact (two-sided) test, which involved a comparison of the numbers of indicated categories for each pair of datasets (**** - p<0.0001).
Figure 7—figure supplement 1
Distribution of RNA:DNA hybrids around the centred origins of PCF and BSF cells.
(A) The profile plots and heatmaps show the distribution of RNA:DNA hybrids (R-loops; Briggs et al., 2018), around the centred origins of PCF and BSF cells (blue line) and the corresponding intergenic shuffled controls (green line; ±2 kb). Mean DRIP-seq signal presents average DRIP-seq signal (Briggs et al., 2018) per 20 bp window. (B) The percentage of origins of PCF and BSF cells and corresponding shuffled controls that overlap with R-loops (overlap calculated without window). The p-values were calculated using Fisher’s exact (two-sided) test, which involved a comparison of the numbers of indicated categories for each pair of datasets (**** - p<0.0001). The mapped origins in the PCF and BSF cells were analysed separately to show that they can be compared with the merged (PCF + BSF) set of origins presented in the Figure 7 and in the main text.
Figure 8
Comparison of stranded SNS-seq origins with MFA-seq replicated regions and TbORC1/CDC6 binding sites.
(A) Chromosomal overview of MFA-seq and stranded SNS-seq origins from Integrative Genomics Viewer (IGV) (scale in Mb). The 11 core chromosomes of the Tb Lister 427–2018 reference genome are shown. The first track shows genes and their orientation (blue presents genes on the plus strand and turquoise presents genes on the minus strand). The second track shows centromeres in red (for chromosomes 9–11 the centromeres are not mapped to the core chromosome) and in grey TSS, TTS, and HT regions 56. Tracks 3–8 show MFA-seq data 42, 43 displaying the ratio of read depth between G2 and S phase in blue (scale 1–1.8). Tracks 3 and 4 show MFA-seq data from PCF cells (track 3 - early S phase to G2 phase ratio and track 4 - late S phase to G2 phase ratio). Tracks 5 and 6 show MFA-seq data from BSF cells (track 5 - early S phase to G2 phase ratio and track 6 - late S phase to G2 phase ratio) 43. Tracks 7 and 8 show MFA tracks from PCF cells. (Track 7 represents the early S phase to G2 phase ratio and track 8 represents the late S phase to G2 phase ratio) 42 (data obtained from Richard McCulloch via personal communication). Track 9 shows ORC1/CDC6 binding sites (retrieved from TriTrypDB) in orange. Track 10 shows the positions of stranded SNS-seq origins in green. (B) Bar plot presenting the percentage of the overlap of stranded SNS-seq origins (in green) and ORC1/CDC6 binding sites (in orange) with MFA-seq regions from the indicated data sets. Overlap was calculated without a window of 100 nt. The first 4 bars are overlap from Devlin et al., 2016 43 data; the last two bars are overlap from Tiengwe et al., 2012 42 data. Overlap of stranded SNS-seq origins with early S phase to G2 phase MFA data sets are shown in light blue and overlap with late S phase to G2 phase MFA data sets are shown in dark blue. (C) Profile plots illustrating the distribution of Orc1/Cdc6 binding sites 42 around centred origins (±2 kb upper panel and ±10 kb lower panel) for merged origins (PCF and BSF) in blue and corresponding shuffled control in light green. The mean Orc1/Cdc6 signal was calculated within 20 bp windows. (D) Profile plots illustrating the distribution of merged origins (PCF and BSF) in blue and corresponding shuffled control in light green around centred TSS (upper panel), TTS (middle panel), and HT regions (lower panel) 56 (±50 kb). The mean origin signal was calculated within 20 bp windows.
Figure 9
A proposed model of origin with the position of different genetic elements and nucleosome occupancy.
Poly(dA) enriched sequences, interspersed with G4 (poly(dA)/G4) are enriched upstream of the origin centres on plus strand and downstream of origin centres on minus strand. Poly(dT) enriched sequences are enriched downstream of origin centres on plus strand and upstream on minus strand. The centre of the origin is a low nucleosome occupancy (LNO) region, flanked by high nucleosome occupancy (HNO) regions. The double arrow lines indicate the position of the summits of the peaks of different origin elements. The presented positions were calculated from the averaged distances. It should be noted that not all origins have the same spacing. The G4 structures, LNO and HNO regions were identified at a limited number of origins; however, they are illustrated on this model to demonstrate the potential of origins to form these structures.
Figure 10
Isolation and validation of short nascent strands for stranded SNS-seq.
(A) Native agarose gel electrophoresis of fractions from sucrose gradients. gDNA was denatured, centrifuged on 5–20% sucrose gradient and fractions were collected from the top (5% sucrose) to the bottom of gradient (20% sucrose). The size of DNA molecular marker is indicated on the right. Fractions containing nucleic acids of 0.5–2.5 kb are encircled. (B) Control of the efficiency of the T4 PNK and λ-exonuclease enzymes. gDNA was fragmented to the size of SNS fibres and 2 µg of starting gDNA was treated once with the indicated number of units of T4 PNK and λ-exonuclease enzymes to check the efficiency of enzymes. The SNS containing fractions were treated under the same condition but 3–4 times. The size of the DNA molecular marker is indicated. (C) RNA is preserved during treatment with T4 PNK and λ-exonuclease enzymes. RNA from the top fractions of the sucrose gradient was treated with the indicated number of units of T4 PNK and λ-exonuclease enzymes to check if the RNA fibres are preserved after this treatment. The size of the DNA molecular marker is indicated.
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Figure 10—source data 1
Original files of the full raw uncropped, unedited gels.
- https://cdn.elifesciences.org/articles/108143/elife-108143-fig10-data1-v1.zip
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Figure 10—source data 2
Figures with the uncropped gels or blots with the relevant bands clearly labelled.
- https://cdn.elifesciences.org/articles/108143/elife-108143-fig10-data2-v1.zip
Author response image 1
Overlap between TbORC1/CDC6-12Myc binding sites (Tiengwe 2012, Cell Reports) and strand‑switch regions (SSRs).
Venn diagram showing the overlap of 990TbORC1/CDC6-12Mycbinding sites (Retrieved from TritrypDB filtered at score 22 to achieve a number of binding sites similar to the one (953 binding sites) published in Tiengwe 2012, Cell Reports) and SSR sites in the genome (Kim 2018, NAR). The intersection shows that 10.3% of Orc1/CDC6 binding sites overlap with 41.8% SSRs. The intersection is subdivided into TSS (orange), TTS in (blue) and HT in (green).
Author response image 2
Metaplots showing the mean nuclesome signal over centred SNS-seq origins in subtelomeric regions.
Two replicates from Maree et al 2019 (PMID: 28344657).
Author response image 3
Distribution of mapped origins in scaled genic and intergenic regions.
Scaled heatmaps present the distribution of the mapped origins and shuffled controls within scaled genic and intergenic regions (± 2 kb).
Additional files
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Supplementary file 1
Numbers of reads.
The numbers of reads obtained after paired-end sequencing, numbers of reads, peaks and mapped origins after different steps of bioinformatic analysis are presented for three biological replicates of PCF and BSF cells. Each replicate contained an SNS-enriched sample and SNS-depleted control. (+) and (-) peaks called present the number of peaks called and localised on the plus or minus DNA strand, respectively. Filtered peaks (+) and (-) represent pairs of peaks with divergent orientation (first (-) followed by (+) peak) (Methods).
- https://cdn.elifesciences.org/articles/108143/elife-108143-supp1-v1.docx
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Supplementary file 2
DNA replication parameters obtained by DNA combing.
DNA replication parameters obtained by DNA combing in two cell types (PCF and BSF) of T. brucei. The estimated numbers of origins were obtained by dividing the genomic sequence length (50.081 Mb) by either the median or the mean IODs calculated on combed fibres.
- https://cdn.elifesciences.org/articles/108143/elife-108143-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/108143/elife-108143-mdarchecklist1-v1.docx
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Stranded short nascent strand sequencing reveals the topology of DNA replication origins in Trypanosoma brucei
eLife 14:RP108143.
https://doi.org/10.7554/eLife.108143.3
