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Old 11-07-2015, 11:39 PM   #1
daanum
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Location: nz

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Default How to detect if the sample is bisulphie converted?

Hi,

I am working with RRBS methylation data. At the moment I have got the sequencing done and have the FAST QC results of all the sequenced samples.

There was one sample during bisulphite conversion process that showed white crystalline precipitate after the incubation of 18 hours after adding bisulphite conversion reagent. I am assuming that might have happened due to incomplete bisulphite conversion of the sample.

Is there any way to check through FASTQC, if the sample is partially bisulphite converted?
Thanks for your help.
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Old 11-08-2015, 10:58 PM   #2
nucacidhunter
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Join Date: Jan 2013
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Default

Per base sequence content in bisulfite converted library read1 will show very low C and high T. However, it would not indicate level of partial conversion. For calculating BS conversion rate one can spike in Lambda DNA.
White precipitate is most likely from imbalance in chemical composition of reaction and for some kits it is normal. For instance, Qiagen kit forms blue precipitate. You may consult your kit manual or supplier tech support to see if it is expected or not and its possible effect on conversion rate.
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