Epigenome-seq state-of-the-art: but is hmC worth the effort for everyone?

A couple of recent papers have demonstrated the ability to distinguish between 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) using modified bisulfite sequencing protocols. These methods are likely to make a real impact on epigenomics when combined with Bis-seq. By sequencing two genomes, Bis-seq for mC and oxBS-seq or TAB-seq for hmC a fuller picture of methylation will emerge. However it is not clear how important biologically the additional data will be or how much it is worth to researchers.

Trying to complete this kind of experiment today on mammalian genomes requires quite a lot of sequencing muscle. Bis-seq depth guidelines are lacking but most people would aim for 50x or greater coverage. This suggests a 100 fold genome coverage per sample, or an expensive experiment. This could leave the approaches as a niche application for people with a strong focus on epigenetics.

oxBS-seq: The University of Cambridge’s method prevents hmC from being protected in a normal bisulfite conversion. 5-hmC is oxidised such that upon bisulfite treatment the bases are converted to uracils. See Quantitative sequencing of 5-methylcytosine and 5-hydroxymethylcytosine at single-base resolution.

TAB-seq: The University of Chicago’s method differs by protecting 5-hmC from conversion. 5-hmC is protected from oxidation by using beta glucosyltransferase whilst 5-mC’s are oxidised such that during bisulfite treatment they behave as if they were not methylated and are converted to uracils. See Base-resolution analysis of 5-hydroxymethylcytosine in the Mammalian genome.

How to make the methods accessible to all: An area that the two methods may have an impact on more quickly is for capture-based studies in cell lines where material is not limiting. It would be possible to create a PCR-free library and perform capture for an exome (or regions of equivalent genomic size) then follow this capture with bisulfite conversion and sequencing. This MethCap-seq (BisCap, oxBS-Cap or TAB-Cap) method could allow larger sample numbers to be run at the depth required without being too expensive.

We have been testing a Nextera based capture prep in my lab which potentially would allow this to be done on very small inputs, however the method is not yet released and requires quite a bit of amplification.

Another alternative would be to combine the methods with the recent Enhanced Reduced representation bisulfite sequencing (ERRBS) published by Maria E. Figueroa’s group in PLoS Genetics.