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DNA methylation is an epigenetic regulator of gene expression with important functions in development and diseases such as cancer. The modified cytosines, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are identified using Illumina sequencing. Libraries are generated using either NEBNext® EM-seq™, an enzyme-based workflow, or by using sodium bisulfite conversion. These methods, however, cannot differentiate between 5mC and 5hmC. Distinguishing these modifications is important as there is increased interest in 5hmCs role in regulating gene expression. Methods currently exist to enable discrimination of 5mC and 5hmC, for example, oxBS-seq and TAB-seq. These methods still rely upon sodium bisulfite conversion and have reduced data quality due to fragmentation and loss of DNA. Here we describe an enzymatic method that enables specific detection of 5hmC. 5hmC is detected using two enzymatic steps. Firstly, 5hmCs are glucosylated, which protects them from subsequent deamination by APOBEC. In contrast, cytosines and 5mCs are deaminated resulting in their conversion to uracil and thymine, respectively. During Illumina sequencing deaminated cytosine and 5mC are read as thymine and the glucosylated 5hmC are read as cytosine. Subtractive analysis of 5hmC data from EM-seq data, which detects both 5mC and 5hmC, permits the identification of individual 5mC and 5hmC sites. 5hmC data were generated for inputs of 0.1 ng to 200 ng DNA isolated from E14 mouse embryonic stem cells and human brain. The 5hmC libraries had similar characteristics to EM-seq libraries, including expected insert sizes due to intact DNA molecules, low duplication rates and minimal GC bias. T4147 phage DNA was used as an internal control, as all cytosines are 5-hydroxymethylated, with 98-99% of cytosines identified as 5hmC. 5mC and 5hmC levels were also profiled during E14 cell differentiation for 10 days. Interestingly, 5hmC levels decreased whereas 5mC levels increased particularly during the first five days of differentiation. LC-MS/MS quantification of this same DNA mirrored the changes observed by sequencing. The ability to discriminate between 5mC and 5hmC will provide key insights into the role of these cytosine modifications in development and disease. Citation Format: Daniel J. Evanich, V. K. Chaithanya Ponnaluri, Vaishnavi Panchapakesa, Ariel Erijman, Matthew A. Campbell, Nan Dai, Bradley W. Langhorst, Romualdas Vaisvila, Louise Williams. Sequencing of 5-hydroxymethylcytosine to single base resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6018. |
