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Old 03-07-2013, 03:51 PM   #1
JIrish
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Default ChIPseq Library Prep Amplification: How many PCR cycles do you use?

I'm currently working to troubleshoot my ChIPseq workflow, and one step where I think I may be having some issues is amplification of my ChIP DNA to create my sequencing library:

We use the Illumina TruSeq ChIP Library Prep Kit, and PippinPrep for gel-purification of the adapter-ligated fragments before PCR.

My concern is the amount of product I end up with after amplification; I would like to minimize amplification bias and avoid over-amplifying my samples, however we have not been able to generate a sufficient amount of DNA (from 10ng of starting material) after amplification, even when running 18 cycles of PCR. I just began making my own library instead of leaving that step to our seq. core. I tried starting the library prep protocol with 100ng of DNA instead of 10ng, and was able to obtain 50uL of ~10nM DNA after 14 cycles of PCR. The Bioanalyzer peak is narrow and the correct size, with no detectable adapter dimers.

While I now have a library for sequencing, I have a gut feeling that I shouldn't need to start with 10 times the amount of DNA spec'd in the protocol to end up with enough DNA in my library after 14 PCR cycles, especially when I read that many people are getting good libraries with only 10-12 cycles (presumably starting with 10ng of ChIP DNA). Also, I won't have the luxury of 100ng starting material for many of my ChIPs.

Could my ligation efficiency be low, causing only a low proportion of my starting material to be amplified? Do these numbers sound reasonable, or is my library prep efficiency low?

Last edited by JIrish; 03-07-2013 at 04:07 PM.
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Old 03-21-2013, 02:16 PM   #2
dsk569
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Default ChIPseq from low amounts of DNA

I have recently heard about this kit from Diagenode.
MicoPlex Library Kit. You can amplify/make Illumina libraries from as low as picogram amounts of DNA. I hope this helps
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Old 03-24-2014, 05:23 AM   #3
bartlosz
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Default Starting material DNA size range

Hi!

We have similar problems and start troubleshooting. We think that our material for chipseq is not fragmented properly - we have some genomic DNA fraction that is poorly fragmented. We will switch right now from Bioru[tor to Covaris, maybe this will help...

Let me know if you find your solution, I will tell you if we have foudn ours...

Best luck,
Bartosz Wojtas
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Old 04-22-2014, 05:44 AM   #4
YanMar
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Location: Sweden

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Default

We were also having trouble with the Illumina TruSeq ChIP library prep kit. Did not work at all and we consistently got very high duplicate read fractions.

We checked the ChIP DNA fragment size saw that the majority of the ChIPed DNA fragments were >500 bp, thus not suitable for Illumina library prep since this protocol requires a size selection step around 250-300 (including the adaptor size!). This, despite that we sheared the chromatin using covaris having ~80% of the fragments between 100-700 bp.

One advice is to check the size of your ChIP DNA to see what size ranges it has. If most of your fragments are >500, the sample is most likely not suitable for Illumina based library prep.
If possible, sonicate the chromatin a bit more before the ChIP, however remember that a long shearing time might disrupt the protein epitope thus impeding proper IP. You might want to check it by WB.

What we did was to change library prep kit!
We are now using the Rubicon ThruPlex library prep kit (itís the same as Diagenodes MicroPlex). The libraries are a bit broader in size but havenít been any problem for our application. Not only is it much faster but size selection is not necessary and itís a single-tube assay so you donít lose so much material like you do when washing and transferring between wells (like with illumina)


Best of luck!
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Old 04-24-2014, 11:06 AM   #5
NextGenSeq
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Default

We found that Diagenode will give high numbers of duplicate reads with low amounts of input ChIP-DNA.

The NuGen Ultralow kit gives fewer duplicate reads and we have gotten it to work with 1 ng or less of ChIP-DNA
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