Hi Pavel,

I did only a little bisulfite sequencing some years ago, but i think the DNA fragmentation is a known problem after the bisulfite treatment. For subsequent PCR amplification and sequencing, you should use primers amlifying sufficiently short sequences to be sure you can obtain a PCR product.

Try to have a look at this paper, maybe it'll help you:

Fumihito Miura, Yusuke Enomoto, Ryo Dairiki, and Takashi Ito

Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging Nucl. Acids Res. first published online May 30, 2012 doi:10.1093/nar/gks454

http://nar.oxfordjournals.org/content/early/2012/05/30/nar.gks454.full

good luck

Hi Pavel,

I did only a little bisulfite sequencing some years ago, but i think the DNA fragmentation is a known problem after the bisulfite treatment. For subsequent PCR amplification and sequencing, you should use primers amlifying sufficiently short sequences to be sure you can obtain a PCR product.

Try to have a look at this paper, maybe it'll help you:

Fumihito Miura, Yusuke Enomoto, Ryo Dairiki, and Takashi Ito

Amplification-free whole-genome bisulfite sequencing by post-bisulfite adaptor tagging Nucl. Acids Res. first published online May 30, 2012 doi:10.1093/nar/gks454

http://nar.oxfordjournals.org/content/early/2012/05/30/nar.gks454.full

good luck

Hi Andrea,

many thanks for your answer. The paper looks interesting.

I've done quite a bit of testing with this and found that chemical denaturation works best, since it is the high temperatures that promote fragmentation. I've also seen differences in fragmentation using different kits (promega, qiagen, epigentek, etc...). I was able to add a chemical denaturation step to kits that use heat denaturation (and remove heat denaturation) without affecting the % conversion too much. You will see an inverse relationship with fragmentation and % conversion, so keep that in mind.

Best, 

Casey