Strange higher size amplicon in ChIP seq Library!!

[Chiper]

Hi, I am seeing a strange amplicon ~2Kb long in all my libraries beside expected ~250-300bp amplicon (picture attached).

I perform ChIP using DNA sheared with bioruptor which peaks ~500bp. I work with low cell number so I get ~5-10 ng after ChIP. Post IP this DNA is further sheared using Covaris and processed for library preparation as per the Solid ChIP-seq library preparation protocol. I used to get ~250-300 bp DNA as expected but in all my recent samples I could see a higher size amplicon ~2 KB beside the expected amplicon. I am not able to find any logical explanation for this!! Has anyone seen this before?

Can I still use these libraries for sequencing after size selection? I am afraid I might introduce bias in my samples. Please help.

[pmiguel]

Your loading dye is tricking you.

The higher molecular weight DNA you see is just the top of a smear of concatamers of your monomer amplicons. The loading dye is blocking signal from the middle section of the smear. Thus the very top of the smear appears to be a band.

Over amplification of your amplicons is said to be a source of such a smear.

--
Phillip

[Chiper]

Dear Phillip,
Thanks for your reply. The band I see is not a smear since I also loaded samples on Bio Analyzer High sensitivity chip and in almost all the samples it is a band around 1.5-2KB region with no DNA in between. In one sample I also saw another faint band around 500 bp.
I amplified the libraries from 12-14 cycles and given the starting amount I had I don't see much on Lonza gel if I amplify less than this. Do you think that concatamers of monomer amplicons can be a source of such band?

[pmiguel]

Dear Phillip,
Thanks for your reply. The band I see is not a smear since I also loaded samples on Bio Analyzer High sensitivity chip and in almost all the samples it is a band around 1.5-2KB region with no DNA in between. In one sample I also saw another faint band around 500 bp.

Ah, well then your loading dye tricked me. I saw the loading dye and just assumed there would be DNA it was hiding.

I amplified the libraries from 12-14 cycles and given the starting amount I had I don't see much on Lonza gel if I amplify less than this. Do you think that concatamers of monomer amplicons can be a source of such band?

Well it does not take much DNA -- for each ng there are 5 billion 200 bp amplicons. So it is better not to over-amplify.

Can you post the high sensitivity chip picture? That would help me decide whether the 2 kb band is a concatamer or something else.

--
Phillip

[Chiper]

Dear Philip,
Sorry for the late reply. Please find the pictures attached.

[pmiguel]

Yes that looks like "overamplification", even though you only did 10-14 cycles. I don't know what causes it, but I remember pictures of the phenomenon from the v1 manual we used during SOLiD fragment library construction training in Foster City.

--
Phillip