Bisulfite Sequencing – Interrogating CpG Methylation at single base pair resolution.

Hi all, just a quick post here.

In previous posts, I have discussed methods of probing DNA methylation with methylation arrays and MeDIP-Chip. Those methods are genome wide but not quite whole genome. The highest resolution method available (and not surprisingly the most expensive and lowest-throughput) is a whole-genome method called bisulfite sequencing.

Bisulfite sequencing is used to map out DNA methylation at single base pair resolution. The technical challenge here is rather apparent – one must be able to differentiate unmethylated DNA from methylated DNA.

To do this, a process called Bisulfite DNA conversion is utilized. DNA is treated with Sodium or potassium bisulfite, this has the effect of converting unmethylated cytosines to uracil, while methylated cytosines (5-methylcytosines) are left intact. During sequencing reactions, uracils are converted to thymine. This makes identifying methylation changes relatively straightforward using bog-standard bioinformatics.

In effect, we are looking at which particular cytosines have been converted to thymines through bisulfite-induced uracil intermediates. To do this, two versions of a reference genome are digitally created Рone with all cytosines converted to thymines, one with all guanines changed to adenines and the resulting sequence is aligned to both, reads that are then uniquely aligned have their previously removed cytosines replaced  and and those cytosines that have been changed to thymine are identified, and these indicate unmethylated cytosines, the rest of the cytosine bases that were subjected to conversion are methylated.

 

Representation of methylation variation as identified by bisulfite sequencing a given locus. (Reference – http://www.springerimages.com/Images/RSS/5-10.1186_1471-2156-10-73-3 )

The costs of this are extremely high, and one may query if whole genome methods are necessary when only a small number of genes are differentially expressed; I, for one, am of the opinion that it may be wise to couple analysis of differentially expressed genes with amplification of targeted regions and bisulfite sequencing on a targeted basis instead of a generalised whole genome search.

Further reading.

[1] Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. http://www.nature.com/nbt/journal/v27/n4/full/nbt.1530.html – example of targeted bisulfite sequencing.

[2] De novo quantitative bisulfite sequencing using the pyrosequencing technology. http://www.sciencedirect.com/science/article/pii/S0003269704004130 – Paper documenting the combination of bisulfite conversion with pyrosequencing, which is ideal for short read length sequencing. (cost per base is high)

[3] A good review of the technique, protocols and associated challenges may be found here – http://www.ncbi.nlm.nih.gov/pubmed/20583099

That’s all from me for this post, a rather short one, I’m afraid.

Cheers,
Exploreable.

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2 responses to “Bisulfite Sequencing – Interrogating CpG Methylation at single base pair resolution.

  1. Thank you for the quick post. I would also like to see bisulfite sequencing performed more along the lines of a gene by gene basis. This would likely involve applications like Sanger sequencing.

    • Indeed. The trouble we have at the moment is that bisulfite sequencing doesn’t really work economically for small sections of genomes; bisulfite pyrosequencing is used for that but it is an absolute pain to carry out.

      With the development of targeted enrichment/amplification by digital PCR though it should be possible to bisulfite sequence individual regions with greater ease.

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