Precise Genomic Mapping of 5-hydroxymethylcytosine via Covalent Tether-directed Sequencing

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by Povilas Gibas, Milda Narmontė, Zdislav Staševskij, Juozas Gordevičius, Saulius Klimašauskas, Edita Kriukienė

5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes, and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high-resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single-nucleotide resolution, named hmTOP-seq (5hmC-specific tethered oligonucleotide–primed sequencing), which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct conjugation chemistries suggested a structural model for the tether-directed nonhomologous polymerase priming enabling theoretical evaluation of suitable tethers at the design stage. The hmT... You can read the full article in the PLOS Biology Blog.

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