Preferential enrichment of AT rich sequences with TruSeq ChIP kit

[Sabine lagger]

Hi all,
I have used the Illumina TruSeq Chip Kit for library preparation and just got the disappointing results.
I get an incredibly high enrichment of AT rich sequences, basically satellite repeats. Unfortunately this happens also in the input samples.
Could it be a bias in the amplification- but why?

Any ideas would be greatly appreciated!!

Thanks,

Sabine

[Jubs]

Hi Sabine,
I also found exactly the same thing, and I'm still puzzled by that. Did you already figure out why, or would have any ideas on what the problem may be?

The only thing I could think of is that these repeats are already somewhat enriched in the ChIP sample (they could be fished out because there may actually have binding sites for the protein of interest; see here http://seqanswers.com/forums/showpos...62&postcount=6) and they would be preferentially amplified in the PCR step of library preparation because they could act as primers(?)

I'm guessing the only way to overcome this would be to have tons of DNA...but when you're working with limited samples that becomes the bottleneck.

Well, I would really appreciate if you, or anyone else here, has any thoughts on this matter.
All the best.

[Sabine lagger]

Hi Jubs,

you are the first person to reply to me about this annoying issue. Finally some relief!
I have done 3 experiments with the Illumina kit and three times this happens to me. Funny enough I have used the Illumina genome wide Truseq kit and there sequencing worked perfectly fine.
The problem is I am doing ChIP from sorted cells and I am very limited with the amounts of DNA I can use for the library prep. We have tried to rule out all kinds of things like contamination from another person in the lab, different polymerases for amplification, etc.
I am still somehow suspecting that something goes wrong with the library amplification. Maybe I am still doing too many cycles with too much material. I am trying a home made library prep method right now- I will let you know if that improves my results.
I am just glad that somebody else experiences the same problem- that seems to rule out a contamination before the library prep.
Please keep me informed if you hear of anything!
Thanks, Sabine

[Jubs]

Hi Sabine,

By Truseq you mean the Truseq Nano? And you wouldn't have enough DNA to stick to that kit?

I was doing some research for PCR-free ways to prepare library from small amounts of DNA and found this kit (http://www.swiftbiosci.com/products/...t-for-illumina), but I haven't tried it yet, or don't know anyone who has.
I also found this paper (http://genomebiology.com/2011/12/2/R18), which you might find useful. The authors have tested several PCR conditions/machines/chemistry to try to reduced bias during library prep.

I sent my samples to a sequencing facility, so library prep was out of my hands... But I checked for enrichment of my regions of interest in the 2 libraries that were prepared from the same sample, and in both I saw that I lost the good stuff. So PCR is preferentially amplifying other things, most likely the satellite repeats that pile up when aligning the reads... What still puzzles me is that AT-rich stuff should be more difficult to amplify, isn't that right?

Maybe you could also test aliquots from each step of your library prep (also aliquots from PCR cyles?) with qPCR to see in which step the bias is being introduced.

Anyway, it would be nice to hear of any progress you might have in your home made prep.

Best luck!