Mapping Retinoic Acid-Dependant 5mC Derivatives in Mouse Embryonic Fibroblasts.

Methylase-assisted bisulfite sequencing (MAB-seq) is a derivatization technique to evaluate the presence of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) at base-pair resolution. Although MAB-seq was originally designed to study these metabolites under steady-state conditions, we have developed an alternative protocol to evaluate the dynamics of 5-fC/5-caC accumulation in response to agonists, such as all-trans retinoic acid (ATRA). In addition, this protocol utilizes a lower quantity of the M.SssI enzyme without compromising methylation efficiency and requires less bench time. Herein, we describe the use of MAB-seq assay to evaluate the generation of 5-fC/5-caC in response to ATRA in mouse embryonic fibroblasts, using the hypermethylated in cancer 1 (Hic1) locus as a model system.

Generative Models for Quantification of DNA Modifications.

There are multiple chemical modifications of cytosine that are important to the regulation and ultimately the functional expression of the genome. To date no single experiment can capture these separate modifications, and integrative experimental designs are needed to fully characterize cytosine methylation and chemical modification. This chapter describes a generative probabilistic model, Lux, for integrative analysis of cytosine methylation and its oxidized variants. Lux simultaneously analyzes partially orthogonal bisulfite sequencing data sets to estimate proportions of different cytosine methylation modifications and estimate multiple cytosine modifications for a single sample by integrating across experimental designs composed of multiple parallel destructive genomic measurements. Lux also considers the variation in measurements introduced by different imperfect experimental steps; the experimental variation can be quantified by using appropriate spike-in controls, allowing Lux to deconvolve the measurements and recover accurately the underlying signal.

A probabilistic generative model for quantification of DNA modifications enables analysis of demethylation pathways.

We present a generative model, Lux, to quantify DNA methylation modifications from any combination of bisulfite sequencing approaches, including reduced, oxidative, TET-assisted, chemical-modification assisted, and methylase-assisted bisulfite sequencing data. Lux models all cytosine modifications (C, 5mC, 5hmC, 5fC, and 5caC) simultaneously together with experimental parameters, including bisulfite conversion and oxidation efficiencies, as well as various chemical labeling and protection steps. We show that Lux improves the quantification and comparison of cytosine modification levels and that Lux can process any oxidized methylcytosine sequencing data sets to quantify all cytosine modifications. Analysis of targeted data from Tet2-knockdown embryonic stem cells and T cells during development demonstrates DNA modification quantification at unprecedented detail, quantifies active demethylation pathways and reveals 5hmC localization in putative regulatory regions.