Second peak on Agilent HS DNA Chip Trace

[mpalermo]

Hi All,

We've been attempting to create an RNA-Seq Library using a Nugen NGS Target prep. The samples are PCR amplified for 15 cycles and cleaned using a Qiagen Gel Band Extraction kit. The samples were run on an Agilent HS DNA chip and we noticed an additional peak following the sample peak around 1000bp. A second gel band extraction was performed and a follow-up HS DNA chip was run...the peaks were still there! We then tried denaturing the samples at 96C for 2 minutes, but it only made the peaks worse.

We've been told that the second peak can be caused by too many PCR cycles - is this what anyone else has found? Has anyone else experienced this/have some insight?

Thank you!

AGTC Staff

[pbluescript]

It is too much PCR. Have a look at one of my previous posts:

http://seqanswers.com/forums/showthread.php?p=64404#post64404

This thread in general should answer your questions.

[mpalermo]

Very helpful, thanks!

[pmiguel]

Hi All,
[...] We then tried denaturing the samples at 96C for 2 minutes, but it only made the peaks worse.

We've been told that the second peak can be caused by too many PCR cycles - is this what anyone else has found? Has anyone else experienced this/have some insight?

Thank you!

AGTC Staff

Actually had not seen a double peak post for a while. Almost a feeling of nostalgia...

Note that your denaturation/renaturation result strongly supports the "bubble product" hypothesis over my "daisy chain" hypothesis. That is, the inserts all have the same sequences flanking them. Thus, kinetically, the most likely outcome are two unrelated library molecules annealing together at their adapters creating a "bubble" in the middle of the amplicon where the unrelated inserts will not anneal.

As to why that would run slower than a completely anneal amplicon of the same length, I have no idea. Or at least for agarose gel electrophoresis, I have no idea. I am pretty sure that single stranded molecules tend to run more slowly on BioAnalyzer chips than double stranded molecule of the same length. So the single stranded region in the middle of the amplicon would tend to cause it to run slower.

But as you probably saw in the other thread, pbluescript shows the double peak phenomenon on agarose gels as well.

--
Phillip

[riehle]

Hi!
I came across the same problem, you were describing in your thread when preparing ChIP-Seq libraries.
What I dont understand is why can't you get rid of this second peak when doing gel out? I cut a 200-300 bp fraction as well as a 300-400 fraction, still when analyzing my library on the Bioanalyzer I see a second peak around 1000 bp.
Can you or someone explain that to me.
Thanxx

Hi All,

We've been attempting to create an RNA-Seq Library using a Nugen NGS Target prep. The samples are PCR amplified for 15 cycles and cleaned using a Qiagen Gel Band Extraction kit. The samples were run on an Agilent HS DNA chip and we noticed an additional peak following the sample peak around 1000bp. A second gel band extraction was performed and a follow-up HS DNA chip was run...the peaks were still there! We then tried denaturing the samples at 96C for 2 minutes, but it only made the peaks worse.

We've been told that the second peak can be caused by too many PCR cycles - is this what anyone else has found? Has anyone else experienced this/have some insight?

Thank you!

AGTC Staff

[pmiguel]

Hi!
I came across the same problem, you were describing in your thread when preparing ChIP-Seq libraries.
What I dont understand is why can't you get rid of this second peak when doing gel out? I cut a 200-300 bp fraction as well as a 300-400 fraction, still when analyzing my library on the Bioanalyzer I see a second peak around 1000 bp.
Can you or someone explain that to me.
Thanxx

If you unintentionally strand-denature your amplicons during isolation from agarose, when they re-anneal, the statistically favored produce will be a bubble product. Bubble products can run at nearly 2x the apparent size of completely double stranded products of the same length. At least on Agilent chips.

During purification of your DNA from agarose, did you use a heating step?

If so, most likely that is the cause.

I should warn you that this double peaking phenomenon is more a curiosity or an annoyance than a real problem. So burning a lot of time on it is probably not warranted.

--
Phillip