OxBS-seq adds an additional oxidative step to bisulfite to discriminate between 5mC and 5hmC. First, 5hmC in genomic DNA is oxidized to 5-formylcytosine (5fC). Unlike 5mC and 5hmC, 5fC is sensitive to deamination by bisulfite, therefore bisulfite treatment of the oxidized DNA converts the 5fC’s to uracil.
Using next-gen sequencing, cytosines that remain in the sequence were 5mC and not 5hmC. A standard bisulfite run is performed in parallel identifying 5mC and 5hmC. By comparing these two sequences, 5hmC presence can be inferred. The major advantage of OxBS-seq over enzymatic approaches such as TAB-seq is that OxBS-seq does not require a highly active TET enzyme which can be expensive and are only about 95% efficient (Yu et al., 2012).
The major drawback of OxBS-seq is the sequencing depth required. Since two runs are preformed and subtracted, error is compounded (Booth et al., 2012). OxBS-seq was recently developed (Booth et al., 2012) and is being used to understand the role of 5hmC in the nervous system (Mellen et al., 2012) and in development (Seisenberger et al., 2012).
OxBS-seq Additional Reading
Booth, M.J., Ost, T.W., Beraldi, D., Bell, N.M., Branco, M.R., Reik, W., and Balasubramanian, S. (2013). Oxidative bisulfite sequencing of 5-methylcytosine and 5-hydroxymethylcytosine. Nat. Protoc. 8, 1841-1851.
This paper is by the same group that developed Ox-BS-seq. The authors go into more depth of the basics of the technique in this paper as well as provide a semi-detailed, easy to follow protocol that covers the important points of the workflow.
This review describes many base modifications with a focus on 5hmC. The authors also describes OxBS-seq in detail and how it compares to TAB-seq.
- Booth, M.J., Branco, M.R., Ficz, G., Oxley, D., Krueger, F., Reik, W., and Balasubramanian, S. (2012). Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution. Science 336, 934-937.
- Mellen, M., Ayata, P., Dewell, S., Kriaucionis, S., and Heintz, N. (2012). MeCP2 binds to 5hmC enriched within active genes and accessible chromatin in the nervous system. Cell 151, 1417-1430.
- Seisenberger, S., Andrews, S., Krueger, F., Arand, J., Walter, J., Santos, F., Popp, C., Thienpont, B., Dean, W., and Reik, W. (2012). The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol. Cell 48, 849-862.
- Yu, M., Hon, G.C., Szulwach, K.E., Song, C.X., Jin, P., Ren, B., and He, C. (2012). Tet-assisted bisulfite sequencing of 5-hydroxymethylcytosine. Nat. Protoc. 7, 2159-2170.