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Old 01-25-2012, 05:26 AM   #1
yog77
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Location: London

Join Date: Jun 2011
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Default Bisulphite Sequencing Proof-reading or NOT proof-reading?

I'm in the process of applying bisulphite PCR amplicon sequencing to the Illumina platform and wondered what peoples thoughts were on whether to use a proof-reading enzyme in the PCR that can read through Uracil's on a bisulphite converted template (I have only come across these two that apparently do):

pfu TURBO Cx Hotstart (Stratagene)
Expand High Fidelity PLUS (Roche)

OR

Do I continue with the standard hotstart Taq we use in the lab for most bisulphite PCR applications which we know to work very well and gives good amplification products (also I have achieved a good optimisation of all my amplicons with this standard Taq).

Some people say that - With the read depth provided by GAII and above, the level of fidelity of standard Taq should not have a major impact on your results. While there is a tenancy to use a proof-reading enzyme in all commercial sequence capture protocols.

Please help - any comments would be grateful
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Old 01-25-2012, 07:45 AM   #2
shawpa
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Default

I recently went through this protocol and used pfu turbo polymerase. I initially tried PCRing with the taq provided in the truseq kit which I believe is phusion polymerase but I could be wrong. That didn't work and I assumed it was because of the U's. But I guess if you have gotten the normal hotstart Taq to work for previous bisulfite products then that wouldn't be an issue for you.
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