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 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 Sequencing abasic sites in DNA at single-nucleotide resolution

2019 ◽  
Vol 11 (7) ◽  
pp. 629-637 ◽  
Author(s):  
Zheng J. Liu ◽  
Sergio Martínez Cuesta ◽  
Pieter van Delft ◽  
Shankar Balasubramanian
Keyword(s):  
Single Nucleotide ◽  
Abasic Sites ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

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 DNAscent v2: detecting replication forks in nanopore sequencing data with deep learning

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Michael A. Boemo
Keyword(s):  
Dna Replication ◽  
High Throughput ◽  
Single Molecule ◽  
Replication Fork ◽  
Genome Stability ◽  
Brdu Incorporation ◽  
Sequencing Data ◽  
Single Nucleotide ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution

Abstract Background Measuring DNA replication dynamics with high throughput and single-molecule resolution is critical for understanding both the basic biology behind how cells replicate their DNA and how DNA replication can be used as a therapeutic target for diseases like cancer. In recent years, the detection of base analogues in Oxford Nanopore Technologies (ONT) sequencing reads has become a promising new method to supersede existing single-molecule methods such as DNA fibre analysis: ONT sequencing yields long reads with high throughput, and sequenced molecules can be mapped to the genome using standard sequence alignment software. Results This paper introduces DNAscent v2, software that uses a residual neural network to achieve fast, accurate detection of the thymidine analogue BrdU with single-nucleotide resolution. DNAscent v2 also comes equipped with an autoencoder that interprets the pattern of BrdU incorporation on each ONT-sequenced molecule into replication fork direction to call the location of replication origins termination sites. DNAscent v2 surpasses previous versions of DNAscent in BrdU calling accuracy, origin calling accuracy, speed, and versatility across different experimental protocols. Unlike NanoMod, DNAscent v2 positively identifies BrdU without the need for sequencing unmodified DNA. Unlike RepNano, DNAscent v2 calls BrdU with single-nucleotide resolution and detects more origins than RepNano from the same sequencing data. DNAscent v2 is open-source and available at https://github.com/MBoemo/DNAscent. Conclusions This paper shows that DNAscent v2 is the new state-of-the-art in the high-throughput, single-molecule detection of replication fork dynamics. These improvements in DNAscent v2 mark an important step towards measuring DNA replication dynamics in large genomes with single-molecule resolution. Looking forward, the increase in accuracy in single-nucleotide resolution BrdU calls will also allow DNAscent v2 to branch out into other areas of genome stability research, particularly the detection of DNA repair.

scholarly journals Chemical probing of the homopurine·homopyrimidine tract in supercoiled DNA at single-nucleotide resolution

FEBS Letters ◽  
1988 ◽  
Vol 234 (2) ◽  
pp. 295-299 ◽  
Author(s):  
M. Vojtíšková ◽  
S. Mirkin ◽  
V. Lyamichev ◽  
O. Voloshin ◽  
M. Frank-Kamenetskii ◽  
...  
Keyword(s):  
Single Nucleotide ◽  
Supercoiled Dna ◽  
Chemical Probing ◽  
Nucleotide Resolution ◽  
Single Nucleotide Resolution
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