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Old 05-15-2012, 02:11 AM   #1
durandg
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Location: Lyon, France

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Red face Shearing of FFPE extracted DNA.

Hello,

I want to perform exome sequencing on a SOLID 5500 with DNA extracted from FFPE.

After a Bioanalyzer run, i've seen -of course- a range of sizes up to Kb.
Even with doing a shearing and a size selection using the standard protocol of Library prep (Life Tech), i select sizes that are bigger that what i usually get, and actually too bog to perform a correct run, for sure.

Does anyone have an idea of bead ratios to use or any program for the Covaris S220 to shear efficiently that kind of degrading DNA

All help will be appreciated :-)

Regards,

Durandg.
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Old 05-16-2012, 09:10 AM   #2
Hamid
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Hi Durandg,

Since you are starting with DNA that is already smaller in size than intact genomic DNA, you can shear down to the size range of interest on your S220, by using the same setting you currently use to shear genomic DNA. The only difference will be that you will have to extend the processing time. My suggestion is that you carry out a time course of up to 50%beyond the time you use for shearing genomic DNA. for example, if your setting requires a 10 minute processing time for genomic DNA, you should try a shearing time course of 12, 14, and 16 minutes.

Thank you

hamid
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Old 05-17-2012, 01:10 AM   #3
durandg
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Hi Hamid,

Thank you a lot! It's really hard to get informations for this kind of samples that are a bit tricky to work with, even on Covaris, Life Tech or other NGS related websites.

I have already made a test and it looks promising! After some adjustments i think it'll be ok.

Thanks again,

D.
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Old 06-26-2013, 08:23 AM   #4
aan
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Hi

Can you share with me the exact covaris conditions you used to fragment DNA isolated from FFPE for exome sequencing? We desire obtain range of 150 - 300 bp.

Thanks
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Old 07-08-2013, 05:41 AM   #5
durandg
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Dear Aan,

Here are the settings we're using, on a Covaris S220; we usually got a peak size at 160bp within a range from 150bp to 180bp.


Peak power 175
Duty factor 10 100s
Cycles/burst 100

These three steps repeated 6 times. This is basically the same program i'm using for blood DNA but with a longer shearing time per cycle (100s versus 60s for blood DNA).

Hope it'll help,
durandg.
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