Subtraction-free and bisulfite-free specific sequencing of 5-methylcytosine and its oxidized derivatives at base resolution

Yibin Liu, Zhiyuan Hu, Jingfei Cheng, Paulina Siejka-Zielińska, Jinfeng Chen, Masato Inoue, Ahmed Ashour Ahmed and Chun-Xiao Song ()
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Yibin Liu: University of Oxford
Zhiyuan Hu: University of Oxford
Jingfei Cheng: University of Oxford
Paulina Siejka-Zielińska: University of Oxford
Jinfeng Chen: University of Oxford
Masato Inoue: University of Oxford
Ahmed Ashour Ahmed: University of Oxford
Chun-Xiao Song: University of Oxford

Nature Communications, 2021, vol. 12, issue 1, 1-8

Abstract: Abstract Although various methods have been developed for sequencing cytosine modifications, it is still challenging for specific and quantitative sequencing of individual modification at base-resolution. For example, to obtain both true 5-methylcytosine (5mC) and true 5-hydroxymethylcytosine (5hmC) information, the two major epigenetic modifications, it usually requires subtraction of two methods, which increases noise and requires high sequencing depth. Recently, we developed TET-assisted pyridine borane sequencing (TAPS) for bisulfite-free direct sequencing of 5mC and 5hmC. Here we demonstrate that two sister methods, TAPSβ and chemical-assisted pyridine borane sequencing (CAPS), can be effectively used for subtraction-free and specific whole-genome sequencing of 5mC and 5hmC, respectively. We also demonstrate pyridine borane sequencing (PS) for whole-genome profiling of 5-formylcytosine and 5-carboxylcytosine, the further oxidized derivatives of 5mC and 5hmC. This work completes the set of versatile borane reduction chemistry-based methods as a comprehensive toolkit for direct and quantitative sequencing of all four cytosine epigenetic modifications.

Date: 2021
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DOI: 10.1038/s41467-021-20920-2

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