Abstract
5‐hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non‐enzymatically in various biological systems. Although 5hmU has been reported to affect biological processes such as protein–DNA interactions, the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report a method to sequence 5hmU at single‐base resolution. We employ chemical oxidation to transform 5hmU to 5‐formyluracil (5fU), followed by the polymerase extension to induce T‐to‐C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing. In combination with the Illumina next generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T‐to‐C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU‐rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNA and thus will contribute to promote the understanding of this modified base.
Highlights
We employ chemical oxidation to transform 5hmU to 5-formyluracil (5fU), followed by the polymerase extension to induce T-to-C base changes owing to the inherent ability of 5fU to form 5fU:G base pairing
In combination with the Illumina generation sequencing technology, we developed polymerase chain reaction (PCR) conditions to amplify the T-to-C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a 5hmU-rich eukaryotic pathogen
We found that the T-to-C percentage change depends on the concentration of dATP during single-extension PCR
Summary
In the control experiment in which no oxidation of the DNA was carried out (that is, 5hmU is not converted to 5fU) the proportion of sequencing reads exhibiting a “C” signal at 5hmU-modified sites were low We applied the method to map 5hmU in the genome of the eukaryotic pathogen Trypanosoma brucei (Figure 3).[14] We mapped 5hmU on chromosome 2, and observed 161 Ts with significant 5hmU signal (0.02 % of all Ts on the chromosome), as defined using a FDR threshold (against “no-oxidation” control) < 0.1.[15,16] As determined using a simulated random distribution, these sites showed significant (p-value = 0.0019) overlap with 5hmU regions obtained using our previously reported chemical enrichment strategy (for details see Supporting Information).[10,13,16]. Arrows indicate the significant sites (FDR < 0.1) [15] and bottom bars are 5hmU regions from enrichment mapping
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