i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks

Biernacka, Anna; Zhu, Yingjie; Skrzypczak, Magdalena; Forey, Romain; Pardo, Benjamín; Grzelak, Marta; Nde, Jules; Mitra, Abhishek; Kudlicki, Andrzej; Crosetto, Nicola; Pasero, Philippe; Rowicka, Maga; Ginalski, Krzysztof

doi:10.1038/s42003-018-0165-9

Cited by 40 publications

(52 citation statements)

References 40 publications

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“…The functional annotations are non-290 random, which is consistent with the hypothesis that the mixture of states actually exists, and that different states may be associated with the cells executing specific transcriptional programs. The possibility of global, coordinated changes in chromatin conformation may have consequences not only for transcriptional programming, but also for genome stability genome instability and DNA repair [22]; such interplay may be confirmed by correlating the inferred states with DNA 295 damage patterns studied at high resolution [23,24]. Finally, the approach may be generalized to use also with diploid nuclei and applied to studying conformation dynamics of the human genome.…”

Section: Resultsmentioning

confidence: 99%

Maximum Parsimony Interpretation of Chromatin Capture Experiments

Homouz

Kudlicki

2019

Preprint

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We present a new approach to inferring global geometric state of chromatin from Hi-C data. Chromatin conformation capture techniques (3C, and its variants: 4C, 5C, Hi-C, etc.) probe the spatial structure of the genome by identifying physical contacts between 30 genomic loci within the nuclear space. In whole-genome conformation capture (Hi-C) experiments, the signal can be interpreted as spatial proximity between genomic loci and physical distances can be estimated from the data. However, the results of these estimations suffer from internal geometric inconsistencies, notoriously violating the triangle inequality. Here we propose that the inconsistencies may be caused not by 35 experimental artifacts but rather by a mixture of cells, each in one of several conformational states, contained in the sample. We have developed and implemented a graph-theoretic approach that identifies the properties of these subpopulations. We show that the geometrical conflicts in a standard yeast HiC dataset, can be explained by only a small number of homogeneous populations of cells (4 populations are sufficient 40 to reconcile 95,000 most prominent impossible triangles, 8 populations can explain 375,000 top geometric conflicts). Finally, we analyze the functional annotations of genes differentially interacting between the populations, suggesting that each inferred subpopulation may be involved in a functionally different transcriptional program. Author Summary 45The global conformation of chromatin within a nucleus plays an important role in regulation of genes. The Hi-C technique can detect proximity between genomic loci, but attempts to use Hi-C data to infer the global conformation lead to hundreds of thousands of impossible geometries, violating the triangle inequality. To date, there was no explanation for this phenomenon. Here, we resolve these discrepancies by modeling 50 the sample as a mixture of nuclei in several conformation states. We have developed a graph-theoretic approach to characterize the shape of chromatin in each of these states. In a real-life situation, as few as 4-5 discrete subpopulations can resolve all spatial inconsistencies in the data. Moreover, the results suggest that each subpopulation is associated with a functionally specific transcriptional program. 55

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Section: Resultsmentioning

confidence: 99%

Maximum Parsimony Interpretation of Chromatin Capture Experiments

Homouz

Kudlicki

2019

Preprint

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“…On the contrary, large scale can detect broad DSBs with low DSB frequency, such as some spontaneous DSBs generated from aberrant replication or transcription 19 . Using enrichment analysis in iSeq package, we identified off-target sites for I-SceI enzyme digestion in an engineered yeast strain (YBP-275) 5 , it shows the ability of iSeq in identifying off-target sites of restriction enzymes.…”

Section: Advantages and Limitationsmentioning

confidence: 99%

“…This deficit has been a direct result of the absence of critical research tools. Recently, several technologies were developed to accurately detect DSBs throughout the genome by sequencing [4][5][6][7][8] , however, there is still lacking advanced statistical methods and tools for analysis and interpretation of these data. Here we present the novel advanced statistical methods implemented as userfriendly software tools that overcome this final challenge.…”

Section: Introductionmentioning

confidence: 99%

“…We developed a method, BLESS, to label DSBs in situ followed by deep sequencing in 2013 4 , which allows the study of DSBs with nucleotide resolution [9][10][11] . Several modifications of our method, such as i-BLESS, END-Seq, Break-Seq or DSB-capture have been published [5][6][7][8] . Working with more than 100 samples from DSB sequencing using these various methods [4][5][6][7][8][9][10][11] , we developed the first dedicated software suite for analysis of DSB sequencing data, especially customized for this unusual, emerging data type.…”

Section: Introductionmentioning

confidence: 99%

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Analyzing and interpreting DNA double-strand break sequencing data

Mitra

Dojer

Fongang

et al. 2020

Preprint

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DNA double-strand breaks (DSBs), are a major threat to genomic stability and may lead to cancer. Several technologies to accurately detect DSBs genome-wide have been developed recently, but still lacking publicly available tools for analysis of the resulting data. Here, we present a step-by-step iSeq package (http://breakome.utmb.edu/software.html), custom designed for analysis and interpretation of DSB-sequencing data. iSeq performs barcode trimming and read counting, and identifies DSB-enriched regions by statistical test and annotate them to the desired genomic features. Applying this package, users can identify and annotate DSB-enriched regions from base pair (eg. Cas9 cleavage sites) up to megabase (eg. DNA replication stress-induced) resolution, and if possible quantify DSB frequencies per cell genome-wide by combining with qDSB-Seq. iSeq can be used for any sequencing-based DSB detection techniques. The analysis for Steps 1-19 can be performed within ~4 hours.Recently, we presented a general method, qDSB-Seq 13 , to quantify DSBs genome-wide, which provides both DSB frequencies per cell and their precise genomic coordinates. qDSB-Seq has been applied to BLESS 4 and i-BLESS 5 , it is also available for END-Seq 6 , Break-Seq 7 , DSBCapture 8 , BLISS 15 , and so on. In this work, we induced spike-in DSBs by a site-specific endonuclease and used these induced DSBs to quantify studied DSBs. qDSB-Seq method has

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“…Our approach relies on inducing spike-in DSBs by a site-specific endonuclease, which are used to quantify DSBs detected by a DSB labeling method e.g. i-BLESS 15 and can be combined with any DSB labeling technique. We present a comprehensive validation and several applications of qDSB-Seq: quantifying DSBs induced by a radiomimetic drug, occurring during replication stress and caused by natural replication fork barriers.…”

Section: Introductionmentioning

confidence: 99%

qDSB-Seq: quantitative DNA double-strand break sequencing

Zhu

Biernacka

Pardo³

et al. 2017

Preprint

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Emerging genome-wide methods for mapping DNA double-strand breaks (DSBs) by sequencing (e.g. BLESS) are limited to measuring relative frequencies of breaks between loci. Knowing the absolute DSB frequency per cell, however, is key to understanding their physiological relevance. Here, we propose quantitative DSB sequencing (qDSBSeq), a method to infer the absolute DSB frequency genome-wide. qDSB-Seq relies on inducing spike-in DSBs by a site-specific endonuclease and estimating the efficiency of the endonuclease cleavage by sequencing or PCR. This spike-in frequency is used to quantify DSB sequencing data. We present validation of the qDSB-Seq method and results of its application. We quantify DSBs resulting from replication stress and the collapse of replication forks on natural fork barriers in the ribosomal DNA. The qDSBSeq approach can be used with any DSB sequencing method and allows accurate comparisons of absolute DSB frequencies across samples and precise quantification of the impact of various DSB-causing agents.peer-reviewed)

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i-BLESS is an ultra-sensitive method for detection of DNA double-strand breaks

Cited by 40 publications

References 40 publications

Maximum Parsimony Interpretation of Chromatin Capture Experiments

Maximum Parsimony Interpretation of Chromatin Capture Experiments

Analyzing and interpreting DNA double-strand break sequencing data

qDSB-Seq: quantitative DNA double-strand break sequencing

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