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 Reproducible inference of transcription factor footprints in ATAC-seq and DNase-seq datasets via protocol-specific bias modeling

2018 ◽  
Author(s):  
Aslihan Karabacak Calviello ◽  
Antje Hirsekorn ◽  
Ricardo Wurmus ◽  
Dilmurat Yusuf ◽  
Uwe Ohler
Keyword(s):  
Transcription Factor ◽  
Open Chromatin ◽  
Specific Sequence ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Distinct Sequence ◽  
Nucleotide Resolution ◽  
The Impact ◽  
Sequence Bias ◽  
Single Nucleotide Resolution

ABSTRACTDNase-seq and ATAC-seq are broadly used methods to assay open chromatin regions genome-wide. The single nucleotide resolution of DNase-seq has been further exploited to infer transcription factor binding sites (TFBS) in regulatory regions via footprinting. Recent studies have demonstrated the sequence bias of DNase I and its adverse effects on footprinting efficiency. However, footprinting and the impact of sequence bias have not been extensively studied for ATAC-seq. Here, we undertake a systematic comparison of the two methods and show that a modification to the ATAC-seq protocol increases its yield and its agreement with DNase-seq data from the same cell line. We demonstrate that the two methods have distinct sequence biases and correct for these protocol-specific biases when performing footprinting. Despite differences in footprint shapes, the locations of the inferred footprints in ATAC-seq and DNase-seq are largely concordant. However, the protocol-specific sequence biases in conjunction with the sequence content of TFBSs impacts the discrimination of footprint from background, which leads to one method outperforming the other for some TFs. Finally, we address the depth required for reproducible identification of open chromatin regions and TF footprints.

scholarly journals Biophysical Principles of Lineage Factor PU.1 Binding Revealed by NextPBMs

2018 ◽  
Author(s):  
Nima Mohaghegh ◽  
David Bray ◽  
Jessica Keenan ◽  
Ashley Penvose ◽  
Kellen K. Andrilenas ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Modes ◽  
Single Nucleotide ◽  
Post Translational Modifications ◽  
Genome Wide ◽  
A Cell ◽  
Nucleotide Resolution ◽  
The Impact ◽  
Single Nucleotide Resolution

ABSTRACTDetermining the biophysical principles that shape transcription factor (TF) binding in a cell-specific manner is key to quantitative models of gene expression. High-throughput (HT) in vitro methods measuring protein-DNA binding are invaluable for relating TF binding affinity to genome-wide binding; however, the impact of cell-specific post-translational modifications (PTMs) and cofactors are not routinely assessed. To address these limitations, we describe a new HT approach, called nextPBMs (nuclear extract protein-binding microarrays), to characterize TF binding that accounts for PTMs and endogenous cofactors. We use nextPBMs to examine the DNA binding of the lineage factor PU.1/Spi1 and IRF8 in human monocytes. We identify two binding modes for PU.1 in monocytes – autonomous binding unaffected by PTMs and cooperative binding with IRF8, and identify a single cooperative mode for IRF8. We characterize the DNA binding of PU.1:IRF8 complexes, and show how nextPBMs can be used to discover cell-specific cofactors and characterize TF cooperativity at single-nucleotide resolution. We show that chromatin state and cofactors both influence the affinity requirements for PU.1 binding sites. Furthermore, we find that the influences of cooperative (IRF8) and collaborative (C/EBPα) cofactors on PU.1-binding-site affinity are independent and additive.

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.

scholarly journals Plants regenerated from tissue culture contain stable epigenome changes in rice

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Hume Stroud ◽  
Bo Ding ◽  
Stacey A Simon ◽  
Suhua Feng ◽  
Maria Bellizzi ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Phenotypic Variability ◽  
Whole Genome ◽  
Single Nucleotide ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Regenerated Plants ◽  
Nucleotide Resolution ◽  
The Impact ◽  
Single Nucleotide Resolution

Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability.

scholarly journals Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

2021 ◽  
Author(s):  
Jane Mellor ◽  
Phillip Lorenz ◽  
Anna Lamstaes ◽  
Harry J Fischl ◽  
Shidong Xi ◽  
...  
Keyword(s):  
Low Cost ◽  
Cost Effective ◽  
Pulse Labelling ◽  
Single Nucleotide ◽  
Functional Regions ◽  
Nascent Rna ◽  
Nucleotide Resolution ◽  
Nascent Transcripts ◽  
The Impact ◽  
Single Nucleotide Resolution

Genomes are pervasively transcribed leading to stable and unstable transcripts that define functional regions of genomes and contribute to cellular phenotypes. Defining comprehensive nascent transcriptomes is pivotal to understand gene regulation, disease processes, and the impact of extracellular signals on cells. However, currently employed methods are laborious, technically challenging and costly. We developed single-nucleotide resolution 4sU-sequencing (SNU-Seq), involving pulse labelling, biotinylation and direct isolation of nascent transcripts. Artificial poly-(A)-tailing of the 3' most nucleotide of nascent transcripts ensures oligo-d(T) primer-based library preparation and sequencing using commercial 3' RNA-Seq kits. We show that SNU-Seq is a cost-effective new method generating even read profiles across transcription units. We used SNU-Seq to identify transcription elongation parameters, to map usage of polyadenylation (PAS) sites and novel enhancers. Remarkably, 4sU labelled nascent RNA accumulates short ~100nt transcripts that map to the 5' end of genes. We show that isolation of these short nascent RNA and sequencing the 5' and 3' ends using size-selected SNU-Seq (ssSNU-Seq) provides highly sensitive annotations of mapped and novel TSSs, promoter-proximal pause/termination sites. Thus, SNU-seq and ssSNU-seq combined yield comprehensive transcriptomics data at low cost with high spatial and temporal resolution.

scholarly journals NET-prism enables RNA polymerase-dedicated transcriptional interrogation at nucleotide resolution

2018 ◽  
Author(s):  
Constantine Mylonas ◽  
Peter Tessarz
Keyword(s):  
Splicing Factors ◽  
Elongation Factors ◽  
Single Nucleotide ◽  
Rna Pol Ii ◽  
Pol Ii ◽  
Genome Wide ◽  
Regulatory Complexity ◽  
Pol Ii Pausing ◽  
Nucleotide Resolution ◽  
The Impact

ABSTRACTThe advent of quantitative approaches that enable interrogation of transcription at single nucleotide resolution has allowed a novel understanding of transcriptional regulation previously undefined. However, little is known, at such high resolution, how transcription factors directly influence RNA Pol II pausing and directionality. To map the impact of transcription/elongation factors on transcription dynamics genome-wide at base pair resolution, we developed an adapted NET-seq protocol called NET-prism (Native Elongating Transcription by Polymerase-Regulated Immunoprecipitants in the Mammalian genome). Application of NET-prism on elongation factors (Spt6, Ssrp1), splicing factors (Sf1), and components of the pre-initiation complex (PIC) (TFIID, and Mediator) reveals their inherent command on transcription dynamics, with regards to directionality and pausing over promoters, splice sites, and enhancers/super-enhancers. NET-prism will be broadly applicable as it exposes transcription factor/Pol II dependent topographic specificity and thus, a new degree of regulatory complexity during gene expression.

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