TABSeq

TAB-seq, short for Tet-assisted Bisulfite Sequencing, is a sequencing method designed to map 5-hydroxymethylcytosine (5hmC) at single-base resolution across the genome. It enables discrimination of 5hmC from unmodified cytosine and from 5-methylcytosine (5mC), providing precise maps of this epigenetic modification in various cell types and tissues.

The method relies on a two-part chemical strategy. First, a glucosyltransferase adds a glucose moiety to 5hmC, producing 5-glucosyl-hydroxymethylcytosine, which protects 5hmC from further chemical alteration. Second, Tet enzymes oxidize 5mC to 5-formylcytosine or 5-carboxylcytosine. After this treatment, bisulfite sequencing is performed: unmodified C and the oxidized forms are deaminated to uracil and read as thymine, while the protected 5hmC remains as cytosine. In the resulting sequencing data, cytosine calls indicate positions that originally contained 5hmC, enabling base-resolution mapping.

Typically, TAB-seq involves preparing a sequencing library from chemically treated DNA and performing high-throughput sequencing, followed by computational analysis to distinguish 5hmC sites from other cytosine states. The technique has been applied to mammalian genomes to study tissue- and development-specific 5hmC landscapes and to compare 5hmC patterns across biological conditions.

Limitations of TAB-seq include the need for precise enzymatic control over glucosylation and Tet oxidation, potential incomplete protection or oxidation, and relatively high input DNA and cost requirements compared with some other methylation assays. Despite these, TAB-seq remains a key method for obtaining locus-specific 5hmC maps at single-base resolution.