Base-Resolution Analysis of Deoxyuridine at the Genome Scale Based on the Artificial Incorporation Modified Nucleobase

Abstract

Deamination of cytosine and dUMP misincorporation have been found to be capable of producing uracil in the genome. This study presents the AI-seq (artificial incorporation modified nucleobase for sequencing), a “base substitution”, which not only is capable of profiling uracil at single-nucleotide resolution and showing its centromeric enrichment but could also reveal that the identified uracil sites are derived from cytosine deamination. All the results indicate the potential biological significance of uracil as the epigenetic modification.

Enzymatic Cleavage-Mediated Extension Stalling Enables Accurate Recognition and Quantification of Locus-Specific Uracil Modification in DNA

“…It has been reported that one uracil modification exists at position Chr1:1369922 (GRCh38) in human genomes . Here, we applied the ECES approach to determine uracil at position Chr1:1369922 of genomic DNA.…”

Assessing and advancing the safety of CRISPR-Cas tools: from DNA to RNA editing

“…CBEs catalyze C-to-dU conversions and finally result in C-to-T transitions 124 . Deoxyuridine (dU) capturing has been well established by NGS-based high-throughput methods such as Excision-seq 153 , dU-seq 154 , UPD-Seq 155 , U-DNA-Seq 156 and AI-seq 157 . By capturing the base editing intermediate dU, a more advanced technique Detect-seq 158 is able to trace both the on-target and off-target editing events of CBE, as recently validated by a similar technique called Ucaps-seq 159 .…”

UdgX-Mediated Uracil Sequencing at Single-Nucleotide Resolution

“…Recently, several UNG-based uracil-sequencing methods have been developed (reviewed in ref 16). They generally utilized UNG to convert uridines to AP sites, followed by chemical labeling 17,18 or incision by an AP endonuclease to generate free 3′OH for further capture. 19−21 Alternatively, an excision-defective UNG-derivate was applied to pull down uracil-containing DNA fragments.…”

“…22 These methods may generate false positives caused by pre-existing DNA strand breaks, AP sites, or other modified nucleotides with aldehyde groups including 5-formyldeoxyuridine (5fU) and 5-formyldeoxycytosine (5fC), although appropriate pretreatment might reduce some interferences. 20,21 In addition, among these methods only Excision-seq 19 and AI-seq 18 Thus, it is limited to genomes with a high density of uracil, e.g., repair-defective S.cerevisiae and E. coli (∼3−8 dUs per 10 3 nucleotides 23,24 ). AI-seq chemically converted UNG-generated AP sites to azide-cytosine and calculated the locations of these modified bases by comparing them to input sequences, making it sensitive to detect dT-derived dUs.…”

“…They generally utilized UNG to convert uridines to AP sites, followed by chemical labeling , or incision by an AP endonuclease to generate free 3′OH for further capture. − Alternatively, an excision-defective UNG-derivate was applied to pull down uracil-containing DNA fragments . These methods may generate false positives caused by pre-existing DNA strand breaks, AP sites, or other modified nucleotides with aldehyde groups including 5-formyl­deoxyuridine (5fU) and 5-formyl­deoxycytosine (5fC), although appropriate pretreatment might reduce some interferences. , In addition, among these methods only Excision-seq and AI-seq can detect dU at base resolution. However, Excision-seq required uracils on both strands which are close to each other to generate proper double-stranded DNA fragments by UNG/Endo IV treatment.…”