I'm hoping that somebody here can explain this unusual result that I'm getting with some pyrosequencing data. I have bisulphite converted a few samples a couple of times over the years for different projects. Recently I was collecting some of this disparate data together and I noticed that when the same pyrosequencing assay was used on these samples, those that were converted later had decreased methylation at the CpG sites of interest compared to the initial bisulphite converted samples. The original conversion was in 2010 and the repeat conversions were in 2014/2015. The assays are the same and it's difficult to blame this on PCR amplifying one clone over another since I've done technical replicates on each aliquot. Even the 2010 converted sample has the same result in 2015 that it did in 2010 but this is different from the 2015 converted sample.

We are using the same kit (Qiagen) for bisulphite conversion and haven't made a major change to our procedure (we follow the Qiagen protocol without modification). Has anybody noticed this phenomenon before? Is it possible that stored DNA would experience methylation changes? It seems more likely that the issue is technical but I'm having trouble figuring out exactly what that technical issue is.

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    $\begingroup$ In experimental troubleshooting questions like these it is difficult to provide an exact answer unless you specify the protocol. You should also add details such as storage conditions, how old was the bisulphite solution etc, level of difference between the two replicates etc. $\endgroup$
    Commented Jun 11, 2015 at 20:37
  • $\begingroup$ Thanks - I added some detail. I unfortunately can't say too much about our pyrosequencing assays until we've published. $\endgroup$
    – Jack
    Commented Jun 12, 2015 at 15:04
  • $\begingroup$ So just to clarify: You have a sample, converted some of it, stored some of it. Sequenced some of the converted sample(A). Wait five years. Sequence some more converted sample(B), convert some of the stored sample, sequence that (C). So A and B match but C is less? $\endgroup$
    – Resonating
    Commented Jun 12, 2015 at 15:26

1 Answer 1


I looked into this and if the effect is real, which it sounds like it is, it may be 5-formylcytosine.

Methylcytosine oxidizes into hydroxymethylcytosine, which reacts with bisulphite exactly like you expect (identically). Hydroxymethylcytosine further oxidizes to 5-formylcytosine, which behaves like unmodified cytosine (i,e. deaminates to form U). TET enzymes mediate this behavior in vivo but I can't find any data on spontaneous methyl group oxidation or what the kinetics might be like. This paper relies on the properties of 5-formylcytosine outlined above.

You could use this paper to test if that's true. Reduction with sodium borohydride converts 5-formylcytosine back to hydroxymethylcytosine, which reads as methylated under bisulfite sequencing.


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