TAB-Seq

TAB-Seq

TAB-Seq is a novel method that uses bisulfite conversion and Tet proteins to study 5hmC . In this protocol, 5hmC is first protected with a glucose moiety that allows selective interaction and subsequent oxidation of 5mC with the Tet proteins. The oxidized genomic DNA is then treated with bisulfite, where 5hmC remains unchanged and is read as a cytosine, while 5mC and unmethylated cytosines are deaminated to uracil and read as thymidines upon sequencing. Deep sequencing of TAB-treated DNA compared with untreated DNA provides accurate representation of 5hmC localization in the genome. 

Pros:

• CpG and non-CpG hydroxymethylation throughout the genome is covered at single-base resolution
• Dense, less dense, and 5hmC in repeat regions are covered
• Method clearly differentiates between 5hmC and 5mC, specifically identifying 5hmC

Cons:

• Bisulfite converts unmethylated cytosines to thymidines, reducing sequence complexity, which can make it difficult to create alignments
• SNPs where a cytosine is converted to thymidine will be missed upon bisulfite conversion
• Requires deep sequencing to provide sufficient depth to cover the entire genome and accurately map the low amounts of 5hmC

• TAB-Seq: Yu M., Hon G. C., Szulwach K. E., Song C. X., Zhang L., et al. (2012) Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome. Cell 149: 1368-1380

1. Gabel H. W., Kinde B., Stroud H., Gilbert C. S., Harmin D. A., et al. (2015) Disruption of DNA-methylation-dependent long gene repression in Rett syndrome. Nature 522: 89-93
2. Guo F., Yan L., Guo H., Li L., Hu B., et al. (2015) The Transcriptome and DNA Methylome Landscapes of Human Primordial Germ Cells. Cell 161: 1437-1452
3. Li Q., Suzuki M., Wendt J., Patterson N., Eichten S. R., et al. (2015) Post-conversion targeted capture of modified cytosines in mammalian and plant genomes. Nucleic Acids Res 43: e81
4. Sun Z., Dai N., Borgaro J. G., Quimby A., Sun D., et al. (2015) A sensitive approach to map genome-wide 5-hydroxymethylcytosine and 5-formylcytosine at single-base resolution. Mol Cell 57: 750-761
5. Nazor K. L., Boland M. J., Bibikova M., Klotzle B., Yu M., et al. (2014) Application of a low cost array-based technique - TAB-Array - for quantifying and mapping both 5mC and 5hmC at single base resolution in human pluripotent stem cells. Genomics 104: 358-367