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DSBCapture: in situ capture and sequencing of DNA breaks

Abstract

Double-strand DNA breaks (DSBs) continuously arise and cause mutations and chromosomal rearrangements. Here, we present DSBCapture, a sequencing-based method that captures DSBs in situ and directly maps these at single-nucleotide resolution, enabling the study of DSB origin. DSBCapture shows substantially increased sensitivity and data yield compared with other methods. Using DSBCapture, we uncovered a striking relationship between DSBs and elevated transcription within nucleosome-depleted chromatin.

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Figure 1: DSBCapture methodology and comparison to BLESS.
Figure 2: Genomic location and epigenetic context of endogenous DSBs in NHEK cells.

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Acknowledgements

We thank G. Legube, LBCMCP, Center for Integrative Biology (CBI), Université de Toulouse, Toulouse, France for providing U2OS AID-DIvA cells. We thank the genomic core facility at the Cancer Research UK Cambridge Institute. R.H.-H. acknowledges EMBO for support (EMBO Long-Term Fellowship to R.H.-H.). We acknowledge support from the University of Cambridge and the Cancer Research UK program. The Balasubramanian laboratory is supported by core funding from Cancer Research UK (C14303/A17197 to S.B.) and by an ERC Advanced Grant (S.B.). S.B. is a senior investigator of the Wellcome Trust.

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Authors

Contributions

S.V.L. developed the DSBCapture method, conceived the study, conducted experiments, interpreted results and wrote the manuscript. G.M. conceived the study, performed bioinformatics analyses, interpreted results and wrote the manuscript. R.H.-H. contributed to the development of the DSBCapture method, conceived the study, conducted experiments, contributed to bioinformatics analyses, interpreted results and wrote the manuscript. E.Y.L. contributed to the development of the DSBCapture method, conceived the study and conducted experiments. D.T. conceived the study, interpreted results and wrote the manuscript. S.B. conceived the study, interpreted results and wrote the manuscript.

Corresponding author

Correspondence to Shankar Balasubramanian.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 DNA processing workflows, adapter sequences and controls.

(a) BLESS DNA processing workflow following the ligation of both proximal and distal adapters. DNA is digested by I-SceI, PCR amplified, digested by XhoI and subsequently subjected to Illumina library preparation, consisting of end repair, size selection (not shown), A-tailing, Illumina adapter ligation and PCR amplification. Large black arrows indicate the site at which sequencing is initiated; the first 11 bases sequenced are shown. (b) DSBCapture DNA processing workflow following the ligation of both modified P5 and P7 Illumina adapters. The DNA is PCR amplified, size selected (not shown), and sequenced. Large black arrow indicates the site at which sequencing is initiated: the first base sequenced identifies the in situ captured break site. (c) Sequences of the modified P5, modified P7 and control modified P5 Illumina adapters as well as DSBCapture PCR primers (forward (PCR F) and reverse (PCR R)). AD identifies the Illumina adapter barcode sequence, three example reverse primers are shown; further primers can be created by substituting the barcode sequence. B = biotin; P = phosphorylated; * = phosphorothioate bond. (d) Orientation of the DSBCapture library on the Illumina flow cell. The first sequencing primer has complementarity to the P5 Illumina adapter and therefore sequencing is initiated from the P5 end. The ligation of the modified P5 Illumina adapter to the DSB in situ enables direct sequencing of the break site in single-end sequencing. (e) Bioanalyser profiles of the DNA products from DSBCapture and BLESS NHEK libraries. DSBCapture: no product is present in the controls performed without T4 DNA ligase during the first (-T4.1) or second (-T4.2) ligation reactions, or in the control performed with the non-biotinylated control modified P5 lllumina adapter (C). A DSBCapture library is only generated when the complete procedure is carried out (+). BLESS: No product is present in the control performed without T4 DNA ligase during the first ligation reaction (-T4.1). The product of BLESS is shown before Illumina library preparation (I-SceI; diluted 1:10) and after Illumina library preparation (Lib).

Supplementary Figure 2 DSBs mapped by DSBCapture at EcoRV and AsiSI restriction sites.

(a) DSBs created by EcoRV cleavage in fixed nuclei, mapped by DSBCapture (n = 1). PCR duplicates have been removed. Data range is shown in square brackets and black boxes illustrate the genomic location of EcoRV sites. A 20 kb region and a 110 bp region are shown. Pink and purple lines: reads from the sense and antisense strand, respectively. As EcoRV is a blunt cutter, reads originate directly from the cleavage site. (b) AsiSI cleavage sites (black boxes) detected by DSBCapture (n = 1). Cleavage by AsiSI generates a 2 bp 3’ overhang; end processing removes this overhang generating the 2 bp gap in the center of the peak. A 2 kb and a 200 bp region are shown.

Supplementary Figure 3 Overlap of DSBs detected by DSBCapture and GUIDE-seq in U2OS cells.

(a) Venn diagram showing the overlap between the DSBCapture peaks and the 25 sites detected by GUIDE-seq4. (b) Genomic tracts showing the 9 DSBs detected by GUIDE-seq that are also detected by DSBCapture. Each panel shows a genomic view of 2,000 bp around the GUIDE-seq detected DSB hotspot. In each panel, from top to bottom: DSBCapture coverage (grey track); peaks detected in DSBCapture by peak calling (black track); GUIDE-seq sites (dark blue track); RefSeq gene track; reference genome sequence (hg19).

Supplementary Figure 4 Analysis of DSBs detected by DSBCapture and BLESS.

(a) Overlap of peaks between two biological replicate experiments for DSBCapture and BLESS. Peaks called in both replicates (high confidence peaks) were used for data analysis. 84,946 and 18,816 high confidence peaks were identified in DSBCapture and BLESS, respectively. (b) Overlap between the high confidence peaks (shown in a) from the BLESS and DSBCapture experiments. The vast majority (98.6 %) of the BLESS peaks are also identified by DSBCapture, whereas 78.2 % of DSBCapture peaks are unique to this method. (c) Venn diagram showing the overlap of DSBs detected as peaks by DSBCapture performed with 50 μg and 20 μg input material. 74,951 peaks are commonly identified by the two conditions (n = 1). (d) Fraction of DSBs with different GC content in the DSBCapture unique peaks divided by the fraction of peaks shared between BLESS and DSBCapture within the same GC content range (fold enrichment). A fold change greater than one represents an increase in DSBs with that particular GC content in the DSBCapture unique peaks. (e) Fold enrichment of DSBCapture peaks with OQs13, calculated as the number of DSBs overlapping to OQs at each indicated % GC sequence content category (x-axis labels) divided by random overlap. All = all 716,311 OQs, irrespectively of GC content; error bars: standard deviation of the fold enrichment over random.

Supplementary Figure 5 Correlation of DSBs with chromatin marks, genic regions and transcription.

(a) Genomic location of DSBs detected by DSBCapture with respect to histone marks H2A.Z, H3K4me3, H3K4me1, H3K27ac, H3K27me3 as well as DNase and POL2B. Two 100 kb genomic regions are shown; upper: a gene dense region on chromosome 19; lower: a region upstream of the EGFR gene. The data range is shown in square brackets, 5’UTRs are highlighted with red boxes. (b) Number and fold enrichment of DSBs at active and inactive enhancers. (c) Fold enrichment of DSBs in genic and intergenic regions over random. Values > 1 indicate that DSBs are preferentially found within that genomic location. Error bars: standard deviation of the fold enrichment over random. (d) Gene expression values measured as rpkm for genes with (left box) or without (right box) DSBs within ± 1 kb of the TSS. Boxes span from the 25th to the 75th percentile with the median marked by a solid bar. All whiskers extend from the 5th to the 95th percentile.

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Supplementary Figures 1–5 and Supplementary Tables 1–4. (PDF 1441 kb)

Supplementary Software

DSBCapture_code_submitted (ZIP 2 kb)

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Lensing, S., Marsico, G., Hänsel-Hertsch, R. et al. DSBCapture: in situ capture and sequencing of DNA breaks. Nat Methods 13, 855–857 (2016). https://doi.org/10.1038/nmeth.3960

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