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bruce01 07-22-2013 04:17 AM

DNA quality for exome sequencing
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Hi all,

I have an exome project upcoming. I sent the sequencing facility 0.8% gel images of the DNA I have extracted (attached for reference). Obviously, some are better than others. But the facility reckon all are not of high enough quality. Nanodrop (I know...) says those that are of decent visual quality (eg 7, 9, 13, 14, 16, 1, 2) are above 200ng/ul, and 260:280 are over 1.8. I extracted with Zymo Miniprep kit.

My question: how long is a piece of string. Or, what sort of quality thresholds are typically used in your lab/facility for exome sequencing. Do all my samples look bad enough not to bother with? I have only ever used DNA for PCR/Sanger sequencing, and thought that at least those samples listed would be ok?

Tissue is liver, samples are from 2009, have been stored -80 since then. RNA seq has been done on them with no issues (ie all RNA samples RINs >8, libraries of good size and quality and QC by KAPA).

Your input is very much appreciated.

kwaraska 07-22-2013 05:58 AM

In my opinion, what matters for exome is that you cover the genome with your DNA and it is not fragmented beyond the size you want to create the library with.

We have used fragmented DNA before to construct libraries.

If the facility you are loking at refuses to work with you, private message me. I work in a core facility where we would work with you.

bruce01 07-22-2013 06:02 AM

@Kwaraska re:fragmentation/degradation, this is what I was thinking. We are going to do post-capture, so the libraries will be made first, including the fragmentation step. So if gDNA is >200bp all should be ok?

From your experience how does the DNA look? Have you made exomic sequence from similar? I know, it's a bit of an ask to compare, but just out of interest?

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