Weird library profile

JustinNelligan

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Join Date: Mar 2013

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#1

Weird library profile

03-11-2013, 02:24 AM

Hello,

We are seeing a weird library profile - double peak + some high molecular weight smear after the range we cut. It's a chip-seq library amplified for 18 cycles - first time we see this specific profile, should we sequence it?

Thanks!
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kbusseq

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Join Date: Mar 2013

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#2

03-25-2013, 09:21 AM

It's possible that the double peak is caused by running too much sample for the chip (I'm guessing your image is from a Bioanalyzer chip or something similar). We've seen some seriously bizarre peaks on our Bioanalyzer (some double like yours and one that was actually a triple peak) that are remedied simply by re-running the sample at a 1:5 or 1:10 dilution.

Sometimes this eliminates all trace of a high MW smear as well, but typically I would guess that the presence of high MW fragments is due to over-amplifying the library. Do you usually amplify for 18 cycles? That is a little high in my experience (which, granted, I don't have tons of yet... ), but if it normally works for you than it might not be the issue.
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