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

2019 ◽  
Author(s):  
Bo Cao ◽  
Xiaolin Wu ◽  
Jieliang Zhou ◽  
Hang Wu ◽  
Michael S. DeMott ◽  
...  
Keyword(s):  
Single Nucleotide ◽  
Strand Breaks ◽  
Dna Structures ◽  
Quantitative Mapping ◽  
Single Strand Breaks ◽  
Dna Modifications ◽  
Nucleotide Resolution ◽  
Generation Sequencing ◽  
Single Nucleotide Resolution ◽  
Quantitative Profiling

AbstractHere we present the Nick-seq platform for quantitative mapping of DNA modifications and damage at single-nucleotide resolution across genomes. Pre-existing breaks are blocked and DNA structures converted to strand-breaks for 3’-extension by nick-translation to produce nuclease-resistant oligonucleotides, and 3’-capture by terminal transferase tailing. Libraries from both products are subjected to next-generation sequencing. Nick-seq is a generally applicable method illustrated with quantitative profiling of single-strand-breaks, phosphorothioate modifications, and DNA oxidation.

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

2020 ◽  
Vol 48 (12) ◽  
pp. 6715-6725 ◽  
Author(s):  
Bo Cao ◽  
Xiaolin Wu ◽  
Jieliang Zhou ◽  
Hang Wu ◽  
Lili Liu ◽  
...  
Keyword(s):  
Genome Stability ◽  
Sublethal Dose ◽  
Cell Phenotype ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Abasic Sites ◽  
Dna Modifications ◽  
Nucleotide Resolution ◽  
Sensitivity Specificity ◽  
Single Nucleotide Resolution

Abstract DNA damage and epigenetic marks are well established to have profound influences on genome stability and cell phenotype, yet there are few technologies to obtain high-resolution genomic maps of the many types of chemical modifications of DNA. Here we present Nick-seq for quantitative, sensitive, and accurate mapping of DNA modifications at single-nucleotide resolution across genomes. Pre-existing breaks are first blocked and DNA modifications are then converted enzymatically or chemically to strand-breaks for both 3′-extension by nick-translation to produce nuclease-resistant oligonucleotides and 3′-terminal transferase tailing. Following library preparation and next generation sequencing, the complementary datasets are mined with a custom workflow to increase sensitivity, specificity and accuracy of the map. The utility of Nick-seq is demonstrated with genomic maps of site-specific endonuclease strand-breaks in purified DNA from Eschericia coli, phosphorothioate epigenetics in Salmonella enterica Cerro 87, and oxidation-induced abasic sites in DNA from E. coli treated with a sublethal dose of hydrogen peroxide. Nick-seq applicability is demonstrated with strategies for >25 types of DNA modification and damage.

scholarly journals Emerging Technologies for Genome-Wide Profiling of DNA Breakage

2021 ◽  
Vol 11 ◽  
Author(s):  
Matthew J. Rybin ◽  
Melina Ramic ◽  
Natalie R. Ricciardi ◽  
Philipp Kapranov ◽  
Claes Wahlestedt ◽  
...  
Keyword(s):  
Genome Instability ◽  
Dna Double Strand Breaks ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Single Strand Breaks ◽  
Genome Wide ◽  
A Genome ◽  
Wide Scale ◽  
Nucleotide Resolution ◽  
Genomic Regions

Genome instability is associated with myriad human diseases and is a well-known feature of both cancer and neurodegenerative disease. Until recently, the ability to assess DNA damage—the principal driver of genome instability—was limited to relatively imprecise methods or restricted to studying predefined genomic regions. Recently, new techniques for detecting DNA double strand breaks (DSBs) and single strand breaks (SSBs) with next-generation sequencing on a genome-wide scale with single nucleotide resolution have emerged. With these new tools, efforts are underway to define the “breakome” in normal aging and disease. Here, we compare the relative strengths and weaknesses of these technologies and their potential application to studying neurodegenerative diseases.

scholarly journals Deciphering the ‘m6A code’ via quantitative profiling of m6A at single-nucleotide resolution

2019 ◽  
Author(s):  
Miguel Angel Garcia-Campos ◽  
Sarit Edelheit ◽  
Ursula Toth ◽  
Ran Shachar ◽  
Ronit Nir ◽  
...  
Keyword(s):  
De Novo ◽  
Critical Role ◽  
Cell Types ◽  
Single Nucleotide ◽  
Sequence Composition ◽  
Disease States ◽  
Extended Sequence ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution ◽  
Quantitative Profiling

AbstractN6-methyladenosine (m6A) is the most abundant modification on mRNA, and is implicated in critical roles in development, physiology and disease. The ability to map m6A using immunoprecipitation-based approaches has played a critical role in dissecting m6A functions and mechanisms of action. Yet, these approaches are of limited specificity, unknown sensitivity, and unable to quantify m6A stoichiometry. These limitations have severely hampered our ability to unravel the factors determining where m6A will be deposited, to which levels (the ‘m6A code’), and to quantitatively profile m6A dynamics across biological systems. Here, we used the RNase MazF, which cleaves specifically at unmethylated RNA sites, to develop MASTER-seq for systematic quantitative profiling of m6A sites at 16-25% of all m6A sites at single nucleotide resolution. We established MASTER-seq for orthogonal validation andde novodetection of m6A sites, and for tracking of m6A dynamics in yeast gametogenesis and in early mammalian differentiation. We discover that antibody-based approaches severely underestimate the number of m6A sites, and that both the presence of m6A and its stoichiometry are ‘hard-coded’ via a simple and predictable code within the extended sequence composition at the methylation sites. This code accounts for ~50% of the variability in methylation levels across sites, allows excellentde novoprediction of methylation sites, and predicts methylation acquisition and loss across evolution. We anticipate that MASTER-seq will pave the path towards a more quantitative investigation of m6A biogenesis and regulation in a wide variety of systems, including diverse cell types, stimuli, subcellular components, and disease states.

scholarly journals CRISPR-BEST: a highly efficient DSB-free base editor for filamentous actinomycetes

2019 ◽  
Author(s):  
Yaojun Tong ◽  
Helene L. Robertsen ◽  
Kai Blin ◽  
Andreas K. Klitgaard ◽  
Tilmann Weber ◽  
...  
Keyword(s):  
Genome Instability ◽  
Cytidine Deaminase ◽  
Double Strand Breaks ◽  
Antimicrobial Compounds ◽  
Dna Double Strand Breaks ◽  
Single Nucleotide ◽  
Strand Breaks ◽  
Genetic Manipulations ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

AbstractFilamentous actinomycetes serve as major producers of various natural products including antimicrobial compounds. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSB) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem). Specifically targeted by an sgRNA, the cytidine deaminase component of CRISPR-BEST efficiently converts C:G to T:A within a window of approximately seven-nucleotides. The system was validated and successfully used in different Streptomyces species.

scholarly journals Characterization of FMR1 Repeat Expansion and Intragenic Variants by Indirect Sequence Capture

2021 ◽  
Vol 12 ◽  
Author(s):  
Valentina Grosso ◽  
Luca Marcolungo ◽  
Simone Maestri ◽  
Massimiliano Alfano ◽  
Denise Lavezzari ◽  
...  
Keyword(s):  
Repeat Expansion ◽  
Single Nucleotide ◽  
Short Read Sequencing ◽  
Sequence Capture ◽  
Long Read ◽  
Repeat Expansions ◽  
Nucleotide Resolution ◽  
Generation Sequencing ◽  
Single Nucleotide Resolution

Traditional methods for the analysis of repeat expansions, which underlie genetic disorders, such as fragile X syndrome (FXS), lack single-nucleotide resolution in repeat analysis and the ability to characterize causative variants outside the repeat array. These drawbacks can be overcome by long-read and short-read sequencing, respectively. However, the routine application of next-generation sequencing in the clinic requires target enrichment, and none of the available methods allows parallel analysis of long-DNA fragments using both sequencing technologies. In this study, we investigated the use of indirect sequence capture (Xdrop technology) coupled to Nanopore and Illumina sequencing to characterize FMR1, the gene responsible of FXS. We achieved the efficient enrichment (> 200×) of large target DNA fragments (~60–80 kbp) encompassing the entire FMR1 gene. The analysis of Xdrop-enriched samples by Nanopore long-read sequencing allowed the complete characterization of repeat lengths in samples with normal, pre-mutation, and full mutation status (> 1 kbp), and correctly identified repeat interruptions relevant for disease prognosis and transmission. Single-nucleotide variants (SNVs) and small insertions/deletions (indels) could be detected in the same samples by Illumina short-read sequencing, completing the mutational testing through the identification of pathogenic variants within the FMR1 gene, when no typical CGG repeat expansion is detected. The study successfully demonstrated the parallel analysis of repeat expansions and SNVs/indels in the FMR1 gene at single-nucleotide resolution by combining Xdrop enrichment with two next-generation sequencing approaches. With the appropriate optimization necessary for the clinical settings, the system could facilitate both the study of genotype–phenotype correlation in FXS and enable a more efficient diagnosis and genetic counseling for patients and their relatives.

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