Agilent Bioanalyzer Negative Peaks?!?

pmiguel · 10-19-2016, 11:27 AM

Here is what I mean:

NegativePeak.png

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 · 10-19-2016, 12:02 PM

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 · 10-19-2016, 01:49 PM

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 · 10-19-2016, 01:55 PM

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 · 10-20-2016, 12:08 PM

Quote:

Originally Posted by 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.

We re-ran the samples, loading 1/4th the amount. Same effect:
NegativePeak2.png

--
Phillip

pmiguel · 10-20-2016, 12:13 PM

Quote:

Originally Posted by 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.

Interesting!

Thanks,
--
Phillip

pmiguel · 10-20-2016, 12:22 PM

Quote:

Originally Posted by 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.

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 · 10-20-2016, 05:31 PM

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 · 10-20-2016, 05:35 PM

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.

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10-19-2016, 11:27 AM	#1
pmiguel Senior Member Location: Purdue University, West Lafayette, Indiana Join Date: Aug 2008 Posts: 2,317	Agilent Bioanalyzer Negative Peaks?!? Here is what I mean: NegativePeak.png 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 Last edited by pmiguel; 10-19-2016 at 11:39 AM.

10-20-2016, 05:31 PM	#8
seqdavis Junior Member Location: Midwest Join Date: Sep 2015 Posts: 5	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?? __________________ SeqDavis

10-19-2016, 12:02 PM	#2
luc Senior Member Location: US Join Date: Dec 2010 Posts: 452	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.

10-19-2016, 01:49 PM	#3
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	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.

10-19-2016, 01:55 PM	#4
RickC7 Member Location: Baton Rouge, Louisiana Join Date: Feb 2010 Posts: 31	Did you check concentration on qubit? May be outside of upper range? Have seen similar, not so pronounced, but diluting helped in our case.

10-20-2016, 05:35 PM	#9
nucacidhunter Jafar Jabbari Location: Melbourne Join Date: Jan 2013 Posts: 1,238	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.