Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage

Cao, Bo; Wu, Xiaolin; Zhou, Jie; Wu, Honglu; Liu, Huan; Zhang, Qinghua; DeMott, Michael S.; Gu, Cheng; Wang, Lianrong; You, Delin; Dedon, Peter C.

doi:10.1093/nar/gkaa473

Cited by 47 publications

(62 citation statements)

References 54 publications

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“…For such applications, though, it is possible that nicks appearing at extremely low penetrance would not be detected using DENT-seq. Furthermore, as recently reported by Cao et al (2020), nick detection methods like DENT-seq could be applied to study other types of DNA damage as well by converting the damage into nicks; for example, base modifications could be studied through treatment with a glycosylase and AP endonuclease from the base excision repair pathway before readout by DENT-seq.…”

Section: Discussionmentioning

confidence: 99%

“…GLOE-seq detects nicks with high resolution by ligation of adapters to free 3 ′ hydroxy (-OH) ends of DNA but, like SSiNGLe, is not specific to nicks and captures DSBs as well (Sriramachandran et al 2020). Finally, Nick-seq is able to identify nicks with single-nucleotide resolution without capturing DSBs but has not yet been applied to gigabase-scale genomes (Cao et al 2020). Descriptions of these methods and the DENTseq method reported here can be found in Supplemental Table S1.…”

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confidence: 99%

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DENT-seq for genome-wide strand-specific identification of DNA single-strand break sites with single-nucleotide resolution

et al. 2020

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DNA single-strand breaks (SSBs), or “nicks,” are the most common form of DNA damage. Oxidative stress, endogenous enzyme activities, and other processes cause tens of thousands of nicks per cell per day. Accumulation of nicks, caused by high rates of occurrence or defects in repair enzymes, has been implicated in multiple diseases. However, improved methods for nick analysis are needed to characterize the mechanisms of these processes and learn how the location and number of nicks affect cells, disease progression, and health outcomes. In addition to natural processes, including DNA repair, leading genome editing technologies rely on nuclease activity, including nick generation, at specific target sites. There is currently a pressing need for methods to study off-target nicking activity genome-wide to evaluate the side effects of emerging genome editing tools on cells and organisms. Here, we developed a new method, DENT-seq, for efficient strand-specific profiling of nicks in complex DNA samples with single-nucleotide resolution and low false-positive rates. DENT-seq produces a single deep sequence data set enriched for reads near nick sites and establishes a readily detectable mutational signal that allows for determination of the nick site and strand with single-base resolution at penetrance as low as one strand per thousand. We apply DENT-seq to profile the off-target activity of the Nb.BsmI nicking endonuclease and an engineered spCas9 nickase. DENT-seq will be useful in exploring the activity of engineered nucleases in genome editing and other biotechnological applications as well as spontaneous and therapeutic-associated strand breaks.

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Section: Discussionmentioning

confidence: 99%

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confidence: 99%

DENT-seq for genome-wide strand-specific identification of DNA single-strand break sites with single-nucleotide resolution

et al. 2020

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“…To date, four NGS-based methods have been developed for the genome-wide mapping of SSBs: SSB-Seq [71,72], Nick-Seq [73], SSiNGLe [74] and GLOE-Seq [75] (Table 1). Although the molecular steps involved in generating sequencing libraries vary, they all rely on the common principle of detecting SSBs by capturing free 3 0 -OH termini.…”

Section: Ngs-based Methods For Genome-wide Mapping Of Ssbsmentioning

confidence: 99%

“…Nick-Seq is perhaps a misnomer for a method designed to detect base lesions or modifications rather than SSBs (Fig. 2B) [73]. In fact, pre-existing 3 0 -OH ends are initially blocked by appending dideoxynucleotides.…”

Section: Nick-seqmentioning

confidence: 99%

Exploring the SSBreakome: genome‐wide mapping of DNA single‐strand breaks by next‐generation sequencing

Zilio

Ulrich

2020

The FEBS Journal

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Mapping the genome-wide distribution of DNA lesions is key to understanding damage signalling and DNA repair in the context of genome and chromatin structure. Analytical tools based on high-throughput next-generation sequencing have revolutionized our progress with such investigations, and numerous methods are now available for various base lesions and modifications as well as for DNA double-strand breaks. Considering that single-strand breaks are by far the most common type of lesion and arise not only from exposure to exogenous DNA-damaging agents, but also as obligatory intermediates of DNA replication, recombination and repair, it is surprising that our insight into their genome-wide patterns, that is the 'SSBreakome', has remained rather obscure until recently, due to a lack of suitable mapping technology. Here we briefly review classical methods for analysing single-strand breaks and discuss and compare in detail a series of recently developed high-resolution approaches for the genome-wide mapping of these lesions, their advantages and limitations and how they have already provided valuable insight into the impact of this type of damage on the genome.

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“…Direct ligation of a sequencing adaptor to the 3’ end of individual DNA strands would be a very attractive means of quantifying DNA damage irrespective of DNA resection, and direct labelling of DNA 3’ ends may reveal replication fork direction, particularly in mutants unable to ligate Okazaki fragments. Some methods aimed at mapping single-strand breaks and base changes theoretically have this capability although they have not been applied to DSBs [34, 35], and very recently the Ulrich lab described such a method, GLOE-seq, that is capable of replication profiling in DNA ligase-deficient yeast and human cells, and also maps DSBs although activity on resected substrates was not tested [36]. Here we describe an alternative method, T ransferase A ctivated E nd L igation sequencing (TrAEL-seq), which accurately maps DNA 3’ ends at DSBs that have undergone DNA resection.…”

Section: Introductionmentioning

confidence: 99%

Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

Kara

Krueger

Rugg‐Gunn

et al. 2020

Preprint

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Understanding the distribution of sites at which replication forks stall, and the ensuing fork processing events, requires genome-wide methods sensitive to both changes in replication fork structure and the formation of recombinogenic DNA ends. Here we describe Tr ansferase- A ctivated E nd L igation seq uencing (TrAEL-seq), a method that captures single stranded DNA 3’ ends genome-wide and with base pair resolution. TrAEL-seq labels DNA breaks, and profiles both stalled and processive replication forks in yeast and mammalian cells. Replication forks stalling at defined barriers and expressed genes are detected by TrAEL-seq with exceptional signal-to-noise, most likely through labelling of DNA 3’ ends exposed during fork reversal. TrAEL-seq also labels unperturbed processive replication forks to yield maps of replication fork direction similar to those obtained by Okazaki fragment sequencing, however TrAEL-seq is performed on asynchronous populations of wild-type cells without incorporation of labels, cell sorting, or biochemical purification of replication intermediates, rendering TrAEL-seq simpler and more widely applicable than existing replication fork direction profiling methods. The specificity of TrAEL-seq for DNA 3’ ends also allows accurate detection of double strand break sites after the initiation of DNA end resection, which we demonstrate by genome-wide mapping of meiotic double strand break hotspots in a dmc1 Δ mutant. Overall, TrAEL-seq provides a flexible and robust methodology with high sensitivity and resolution for studying DNA replication and repair, which will be of significant use in determining mechanisms of genome instability.

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Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage

Cited by 47 publications

References 54 publications

DENT-seq for genome-wide strand-specific identification of DNA single-strand break sites with single-nucleotide resolution

DENT-seq for genome-wide strand-specific identification of DNA single-strand break sites with single-nucleotide resolution

Exploring the SSBreakome: genome‐wide mapping of DNA single‐strand breaks by next‐generation sequencing

Genome-wide analysis of DNA replication and DNA double strand breaks by TrAEL-seq

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