Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq

Abstract

A complete understanding of the function of Ten-eleven translocation (TET) family of dioxygenase-mediated DNA demethylation requires new methods to quantitatively map oxidized 5-methylcytosine (5mC) bases at high-resolution. We have recently developed a methylase-assisted bisulfite sequencing (MAB-seq) method that allows base-resolution mapping of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), two oxidized 5mC bases indicative of active DNA demethylation events. In standard bisulfite sequencing (BS-seq), unmodified C, 5fC and 5caC are read as thymine. Thus 5fC/5caC cannot be distinguished from C; in MAB-seq, unmodified C is enzymatically converted to 5mC, allowing direct mapping of rare modifications such as 5fC and 5caC. By combining MAB-seq with chemical reduction of 5fC to 5hmC, we also developed caMAB-seq, a method for direct 5caC mapping. Compared to subtraction-based mapping methods, MAB-seq and caMAB-seq require less sequencing efforts and enable robust statistical calling of 5fC/5caC. MAB-seq and caMAB-seq can be adapted to map 5fC/5caC at the scale of whole genome (WG-MAB-seq), within specific genomic regions enriched for enhancer-marking histone modifications (ChIP-MAB-seq), or at CpG-rich sequences (reduce-reprepsentation (RR)-MAB-seq) such as gene promoters. The full protocol, including DNA preparation, enzymatic treatment, library preparation and sequencing, can be completed within 6-8 d.

methyl-ATAC-seq measures DNA methylation at accessible chromatin

“…Our protocol for mATAC-seq can potentially be integrated with existing methods for combinatorial detection of other DNA modifications including 5-hydroxymethylcytosine (Yu et al 2012;Booth et al 2013), 5-formylcytosine (Song et al 2013), and 5-carboxylcytosine (Wu et al 2016). ChIPmentation uses Tn5 tagmentation in a chromatin immunoprecipitation workflow (Schmidl et al 2015).…”

Simultaneous mapping of active DNA demethylation and sister chromatid exchange in single cells

“…S1A). Because M.SssI treatment is carried out in normal enzymatic conditions (Wu et al 2016), it is possible that M.SssI treatment can be combined with other steps of library preparation without introducing additional rounds of DNA purification. Currently, two different library preparation strategies, reduced representation BS-seq (RRBS) (Meissner et al 2005) and post-bisulfite adaptor tagging (PBAT) (Miura et al 2012), have been implemented to perform low-input or single-cell BS-seq (Guo et al 2013;Smallwood et al 2014;Farlik et al 2015).…”

Invited Review – A 5‐year update on epigenome‐wide association studies of DNA modifications in Alzheimer’s disease: progress, practicalities and promise

“…However, these methods do not quantify other cytosine modifications, namely 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) (see Box 1), where their functional relevance is controversial as they are known to be very lowly expressed in brain tissue [55]. Aside from long-read third-generation sequencing, methylase-assisted bisulfite sequencing (MAB-seq) is another method that allows base resolution mapping of 5fC and 5caC, by enzymatically converting unmodified cytosine to 5mC [56]. A recent study used this technique alongside oxidative bisulfite sequencing in induced pluripotent stem cells (iPSCs) models of AD to allow the simultaneous quantification of four cytosine states (5mC, 5hmC, 5fC/5caC and unmodified cytosine) [57].…”