Chemical Modification-Assisted Bisulfite Sequencing (CAB-Seq) for 5-Carboxylcytosine Detection in DNA

Abstract

5-Methylcytosine (5mC) in DNA can be oxidized stepwise to 5-hydroxymethylcytosine (5hmC), 5- formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the TET family proteins. Thymine DNA glycosylase can further remove 5fC and 5caC, connecting 5mC oxidation with active DNA demethylation. Here we present a chemical modification-assisted bisulfite sequencing (CAB-Seq) that can detect 5caC with single-base resolution in DNA. We optimized 1-ethyl-3- [3-dimethylaminopropyl]carbodiimide hydrochloride (EDC)- catalyzed amide bond formation between the carboxyl group of 5caC and a primary amine group. We found that the modified 5caC can survive the bisulfite treatment without deamination. Therefore, this chemical labeling coupled with bisulfite treatment provides a base-resolution detection and sequencing method for 5caC.

Base-resolution maps of 5-formylcytosine and 5-carboxylcytosine reveal genome-wide DNA demethylation dynamics

“…We have demonstrated that chemical modification-assisted bisulfite sequencing (CAB-seq) can detect the base-resolution information of 5fC and 5caC [10,13]. However, due to their low abundance, a direct, whole-genome bisulfite sequencing is impractical.…”

“…However, due to their low abundance, a direct, whole-genome bisulfite sequencing is impractical. We present here a pre-enrichment-based bisulfite sequencing strategy, or DNA immunoprecipitation-coupled CAB-seq (DIP-CAB-seq; Figure 1A and Supplementary information, Figure S1A), to generate the genome-wide, single-base resolution maps for 5fC and 5caC [10,13,14].…”

Analysis of DNA modifications in aging research

“…RedBS-seq was developed to measure levels of fmC through reducing the fmC base back to hmC in order for quantify by sequencing, much like the Ox-BS method (Booth et al 2014). caC can be quantified using CAB-seq, where EDC is used to chemically modify the 5-caC to prevent it from converting during bisulfite conversion, and levels quantified by sequencing (Lu et al 2013). An alternative is methylation assisted bisulfite sequencing (MAB-Seq) which can be used to determine, in a base-by-base fashion combined caC and fC (Neri et al 2016).…”

Current technological advances in mapping new DNA modifications

“…This chemical conversion makes 5caC resistant to bisulfite-driven deamination, allowing 5caC mapping after bisulfite-sequencing by comparison between protected versus unprotected DNA. Importantly, the EDC-coupling reaction of 5caC does not introduce bias during PCR amplification [54]. This recently optimised technique has not been used so far to map 5caC in the mammalian genome.…”