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

2020 ◽  
Vol 31 (1) ◽  
pp. 75-87
Author(s):  
Joshua J. Elacqua ◽  
Navpreet Ranu ◽  
Sarah E. DiIorio ◽  
Paul C. Blainey
Keyword(s):  
Strand Break ◽  
Single Strand ◽  
Single Nucleotide ◽  
Single Strand Break ◽  
Specific Identification ◽  
Genome Wide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

scholarly journals NickSeq for genome-wide strand-specific identification of DNA single-strand break sites with single nucleotide resolution

2019 ◽  
Author(s):  
Joshua J. Elacqua ◽  
Navpreet Ranu ◽  
Sarah E. Dilorio ◽  
Paul C. Blainey
Keyword(s):  
Genome Editing ◽  
Nuclease Activity ◽  
Single Strand ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Single Strand Break ◽  
Genome Wide ◽  
Nucleotide Resolution ◽  
The Impact ◽  
Single Nucleotide Resolution

ABSTRACTDNA single-strand breaks (SSBs), or ‘nicks’, are the most common form of DNA damage. Nicks occur at rates of tens of thousands per cell per day, and result from many sources including oxidative stress and endogenous enzyme activities. Accumulation of nicks, due to high rates of occurrence or defects in repair enzymes, has been implicated in multiple diseases. However, improved methods for nick analysis are needed to learn how their locations and number 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 target sites. There is currently a pressing need for methods to study unintended nicking activity genome-wide to evaluate the impact of emerging genome editing tools on cells and organisms. Here we developed a new method, NickSeq, for efficient strand-specific profiling of nicks in complex DNA samples with single nucleotide resolution and low false-positive rates. NickSeq produces deep sequence datasets enriched for reads near nick sites and establishes a readily detectable mutational signal that allows for determination of the nick site and strand. In this work, we apply NickSeq to profile off-target activity of the Nb.BsmI nicking endonuclease and an engineered spCas9 nickase. NickSeq will be useful in exploring the relevance of spontaneously occurring or repair-induced DNA breaks in human disease, DNA breaks caused by DNA damaging agents including therapeutics, and the activity of engineered nucleases in genome editing and other biotechnological applications.

scholarly journals Mapping ribonucleotides embedded in genomic DNA to single-nucleotide resolution using Ribose-Map

2020 ◽  
Author(s):  
Alli L. Gombolay ◽  
Francesca Storici
Keyword(s):  
Computing Time ◽  
Wide Distribution ◽  
Sequencing Analysis ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Hands On ◽  
Nucleotide Resolution ◽  
User Friendly ◽  
Single Nucleotide Resolution

ABSTRACTRibose-Map is a user-friendly, standardized bioinformatics toolkit for the comprehensive analysis of ribonucleotide sequencing experiments. It allows researchers to map the locations of ribonucleotides in DNA to single-nucleotide resolution and identify biological signatures of ribonucleotide incorporation. In addition, it can be applied to data generated using any currently available high-throughput ribonucleotide sequencing technique, thus standardizing the analysis of ribonucleotide sequencing experiments and allowing direct comparisons of results. This protocol describes in detail how to use Ribose-Map to analyze raw ribonucleotide sequencing data, including preparing the reads for analysis, locating the genomic coordinates of ribonucleotides, exploring the genome-wide distribution of ribonucleotides, determining the nucleotide sequence context of ribonucleotides, and identifying hotspots of ribonucleotide incorporation. Ribose-Map does not require background knowledge of ribonucleotide sequencing analysis and assumes only basic command-line skills. The protocol requires less than 3 hr of computing time for most datasets and about 30 min of hands-on time.

scholarly journals Single-nucleotide resolution dynamic repair maps of UV damage in Saccharomyces cerevisiae genome

2018 ◽  
Vol 115 (15) ◽  
pp. E3408-E3415 ◽  
Author(s):  
Wentao Li ◽  
Ogun Adebali ◽  
Yanyan Yang ◽  
Christopher P. Selby ◽  
Aziz Sancar
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Excision Repair ◽  
Model Organism ◽  
Early Time ◽  
Uv Damage ◽  
Single Nucleotide ◽  
Pyrimidine Dimers ◽  
Genome Wide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

We have adapted the eXcision Repair-sequencing (XR-seq) method to generate single-nucleotide resolution dynamic repair maps of UV-induced cyclobutane pyrimidine dimers and (6-4) pyrimidine–pyrimidone photoproducts in the Saccharomyces cerevisiae genome. We find that these photoproducts are removed from the genome primarily by incisions 13–18 nucleotides 5′ and 6–7 nucleotides 3′ to the UV damage that generate 21- to 27-nt-long excision products. Analyses of the excision repair kinetics both in single genes and at the genome-wide level reveal strong transcription-coupled repair of the transcribed strand at early time points followed by predominantly nontranscribed strand repair at later stages. We have also characterized the excision repair level as a function of the transcription level. The availability of high-resolution and dynamic repair maps should aid in future repair and mutagenesis studies in this model organism.

scholarly journals CNCC: an analysis tool to determine genome-wide DNA break end structure at single-nucleotide resolution

BMC Genomics ◽  
2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Karol Szlachta ◽  
Heather M. Raimer ◽  
Laurey D. Comeau ◽  
Yuh-Hwa Wang
Keyword(s):  
Mapping Technique ◽  
Nucleotide Position ◽  
Analysis Tool ◽  
Single Nucleotide ◽  
Genome Wide ◽  
A Genome ◽  
Cross Correlation Analysis ◽  
Dna Break ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

Abstract Background DNA double-stranded breaks (DSBs) are potentially deleterious events in a cell. The end structures (blunt, 3′- and 5′-overhangs) at DSB sites contribute to the fate of their repair and provide critical information concerning the consequences of the damage. Therefore, there has been a recent eruption of DNA break mapping and sequencing methods that aim to map at single-nucleotide resolution where breaks are generated genome-wide. These methods provide high resolution data for the location of DSBs, which can encode the type of end-structure present at these breaks. However, genome-wide analysis of the resulting end structures has not been investigated following these sequencing methods. Results To address this analysis gap, we develop the use of a coverage-normalized cross correlation analysis (CNCC) to process the high-precision genome-wide break mapping data, and determine genome-wide break end structure distributions at single-nucleotide resolution. We take advantage of the single-nucleotide position and the knowledge of strandness from every mapped break to analyze the relative shifts between positive and negative strand encoded break nucleotides. By applying CNCC we can identify the most abundant end structures captured by a break mapping technique, and further can make comparisons between different samples and treatments. We validate our analysis with restriction enzyme digestions of genomic DNA and establish the sensitivity of the analysis using end structures that only exist as a minor fraction of total breaks. Finally, we demonstrate the versatility of our analysis by applying CNCC to the breaks resulting after treatment with etoposide and study the variety of resulting end structures. Conclusion For the first time, on a genome-wide scale, our analysis revealed the increase in the 5′ to 3′ end resection following etoposide treatment, and the global progression of the resection. Furthermore, our method distinguished the change in the pattern of DSB end structure with increasing doses of the drug. The ability of this method to determine DNA break end structures without a priori knowledge of break sequences or genomic position should have broad applications in understanding genome instability.

DSBCapture: in situ single-nucleotide resolution DNA double-strand break mapping

2016 ◽  
Author(s):  
Stefanie Lensing ◽  
Stefanie Lensing ◽  
Giovanni Marsico ◽  
Robert Hänsel-Hertsch ◽  
Enid Lam ◽  
...  
Keyword(s):  
Strand Break ◽  
Double Strand Break ◽  
Single Nucleotide ◽  
Dna Double Strand Break ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution
Sign in / Sign up

Export Citation Format

Share Document