Agilent Bioanalyzer Negative Peaks?!?

[pmiguel]

Here is what I mean:



This is some input DNA obtained by reverse-crosslinking sonicated chromatin submitted by someone interested in our doing library construction for them. It was run on an Agilent Bioanalyzer High Sensitivity Chip.

Everything looks great except -- the peak produced has negative amplitude!
The Agilent Bioanalyzer Trouble shooting manual suggests this could happen as a result of the sample containing "detergents" or "dyes".

Anyone seen this and been able to figure out the cause?

(Yes, I know I answered the post of someone asking nearly the same question 4 years ago. But having seen this a few time recently in our lab, I'm finding all the responses in that thread, including my own, unhelpful.)

--
Phillip

[luc]

We have seen this before (slightly less dramatic) also with ChIP DNA from customers. Perhaps there is a reagent in the Diagenode ChIP kits that can have this effect? The library prep and sequencing went well nevertheless.

[nucacidhunter]

I have seen it when DNA is eluted with buffer containing 0.1% Tween 20 even though in the Chip it is further diluted. Running the same sample on Screentape HS5000 is OK.

[RickC7]

Did you check concentration on qubit? May be outside of upper range? Have seen similar, not so pronounced, but diluting helped in our case.

[pmiguel]

Did you check concentration on qubit? May be outside of upper range? Have seen similar, not so pronounced, but diluting helped in our case.

We re-ran the samples, loading 1/4th the amount. Same effect:


--
Phillip

[pmiguel]

I have seen it when DNA is eluted with buffer containing 0.1% Tween 20 even though in the Chip it is further diluted. Running the same sample on Screentape HS5000 is OK.

Interesting!

Thanks,
--
Phillip

[pmiguel]

I have seen it when DNA is eluted with buffer containing 0.1% Tween 20 even though in the Chip it is further diluted. Running the same sample on Screentape HS5000 is OK.

Alas, after checking with the people doing the sample prep, they don't use Tween. They use SDS. They also do a minelute clean-up afterwards.

--
Phillip

[seqdavis]

Phillip,

Have you checked this resource from Agilent - slide 12??

http://www.agilent.com/cs/library/sl...ng_Feb2014.pdf

Maybe the sample needs one more clean up??

[nucacidhunter]

This suggests multiple causes including detergents and possibly chaotropic salts remains from column purification. Probably columns need to be washed more times than manufacturer recommends.