Quantitative sequencing of 5-formylcytosine in DNA at single-base resolution

• Published in: Nature chemistry Vol. 6; no. 5; pp. 435 - 440
• Main Authors: Booth, Michael J., Marsico, Giovanni, Bachman, Martin, Beraldi, Dario, Balasubramanian, Shankar
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 01-05-2014; Nature Publishing Group
• Subjects: 639/638/92/147; Analytical Chemistry; Animals; Biochemistry; Chemical reduction; Chemistry; Chemistry/Food Science; Cytosine - analogs & derivatives; Cytosine - analysis; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - genetics; DNA methylation; Embryonic Stem Cells - metabolism; Genomes; Genomics; Inorganic Chemistry; Mammals; Mice; Organic Chemistry; Oxidation; Physical Chemistry; Sodium; Stem cells; Sulfites; United Kingdom > UK
• ISSN: 1755-4330; 1755-4349
• DOI: 10.1038/nchem.1893

Recently, the cytosine modifications 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) were found to exist in the genomic deoxyribonucleic acid (DNA) of a wide range of mammalian cell types. It is now important to understand their role in normal biological function and disease. Here we introduce reduced bisulfite sequencing (redBS-Seq), a quantitative method to decode 5fC in DNA at single-base resolution, based on a selective chemical reduction of 5fC to 5hmC followed by bisulfite treatment. After extensive validation on synthetic and genomic DNA, we combined redBS-Seq and oxidative bisulfite sequencing (oxBS-Seq) to generate the first combined genomic map of 5-methylcytosine, 5hmC and 5fC in mouse embryonic stem cells. Our experiments revealed that in certain genomic locations 5fC is present at comparable levels to 5hmC and 5mC. The combination of these chemical methods can quantify and precisely map these three cytosine derivatives in the genome and will help provide insights into their function.
Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Reduced bisulfite sequencing is now developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells.