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Old 06-10-2011, 05:33 AM   #1
ETHANol
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Default ChIP-seq library prep Illumina Kapa

I prepared a couple ChIP-seq libraries using the exact protocol from the Illumina ChIP-seq library prep kit. Except at the last step of library enrichment by PCR I dived each sample in half. Half I amplified with Phusion polymerase and the other half I amplified with the Kapa Biosysmtems HiFi Library Amplification polymerase.

The cycling conditions were as follows:
Initial denaturation: 45 sec 98˚ C
18 cycles of: 15 sec 98˚ C
30 sec 65˚ C
30 sec 72˚ C
Final extension: 1 min 72˚ C

1 ul of product was run on the Agilent Bioanalyzer (DNA 1000 kit).

With the Kapa Biosystems polymerase all the DNA is up between 500 and 700 bp.

With Phusion polymerase most of the material is in the expected range between 200 and 300 bp. Although there is some up in the 500 to 700 bp range.

The starting material was digested with Micrococcal Nuclease so that about 80% of the DNA was mono-nucleosomal (145bp). Before library amplification the DNA was gel purified according the the instructions of the Illumina kit.

What is causing the large DNA fragments? How can it be minimized?

Kapa technical services says it is probably due to over amplification. Why would over amplification cause the fragments to double in size?

Anybody else using Kapa HiFi polymerase for ChIP-seq library amplification? How many cycles are you using?

Any suggestions greatly appreciated.
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Old 06-10-2011, 05:35 AM   #2
ETHANol
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Oops, I forgot to add image of the Bioanalyzer output. Here as .pdf.
Sample #1 is the input and Sample #2 is the ChIP.
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File Type: pdf mef_chip_bioanalyzer.pdf (54.5 KB, 273 views)
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Old 06-10-2011, 06:18 AM   #3
ETHANol
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I just got a much more convincing answer from Kapa. I'll leave this post here since it may be useful to others.

This is what Kapa says:
"We've had feedback from one user (amplifying TruSeq libraries) who found that when using the same number of cycles with KAPA HiFi as previously optimized for with Phusion, double-sized products were formed. I've attached a screenshot of the BioAnalyzer plot showing these results. Reducing the number of cycles (from 18 to 10) resulted in correctly sized products.

As far as how many cycles to use - In this example, a portion of the 18 cycle PCR products is still correctly sized. In your example, larger products are present in the Phusion sample (albeit at a lower level), and a very small portion of the PCR products generated with KAPA HiFi is correctly sized. To me, this would indicate that reducing the number of cycles used with KAPA HiFi to 10 cycles or less should result in mostly correctly-sized products. Of course, this is just an estimate and some validation in your workflow will unfortunately have to be done.

Our theory is that once the primers and dNTPs are depleted, DNA self-anneals and forms hybridized products.

We have a product which may be of interest to you if over-amplification is a concern. It's a real-time Library Amplification kit, containing a high-fidelity real-time PCR mix (very similar to KAPA HiFi HotStart ReadyMix, so all the same bias-reducing benefits apply) and a set of four fluorescent standards. During amplification on a real-time PCR instrument, the fluorescence of the sample is monitored, and once it reaches the appropriate fluorescent standard (typically std 2), amplification is terminated. I've attached the data sheet for this product.

I hope this information is helpful. If you have any questions or comments, please do not hesitate to contact us.

Thanks and kind regards,"

The good news is less cycles is better. 10 cycles vs. 18 is a huge difference. Looks like we can go down a lot. The bad news is some optimization needs to be done. I guess the best thing would be to use the real-time kit but we already bought the standard.

Attached is the image they sent me.
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File Type: jpg Overamplification_example.jpg (35.2 KB, 152 views)
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Old 11-19-2012, 11:42 AM   #4
kwaraska
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Default Our experience with Kapa

We use the Kapa and had to drop to 10 cycles for all applications.

Hopefully doing so will prevent further problems.
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Old 10-24-2013, 01:15 PM   #5
tman
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Default Could be bioanalyzer artifact

This happens when you overload the bioanalyzer (get clumps at the higher MW end).
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Old 11-05-2013, 01:12 PM   #6
Genohub
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What about heating an aliquot of those higher MW libraries to 95 deg, and then snap cooling, followed by a run on an RNA chip. If you get back a single peak, the libraries may be usable.

- Genohub
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Old 11-12-2014, 01:22 AM   #7
EmiVig
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Hi Genohub,
I had the same problem with HMW band...as soon as I did 4min at 95C and snap cooling, the HMW band disappeared and I got back my nice 250-300bp! Do you think that this library might still be usable for NGS? Do you think that standardizing my LIBprep protocol adding this denaturation step after the PCR enrichment would be ok for sequencing?
Thanks a lot for your help!
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Old 01-21-2015, 05:34 PM   #8
Genohub
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Yes, you can use those libraries for sequencing. It likely doesn't need to be part of your standard procedure. The HMW bands tend to appear with over amplification or when there is a depletion of PCR primers. With the right input / PCR titration, you should be able to avoid these higher bands.

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Old 01-26-2015, 10:28 AM   #9
NextGenSeq
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It is a heteroduplex. We use 12 to 14 cycles when using KAPA HiFi polymerase since it gives higher yields.
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Old 01-29-2015, 03:09 PM   #10
kerplunk412
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The high MW products are likely what I call "bubble products." This is caused when primers become depleted and products begin to anneal to one another that are only complementary on the adapters, forming a dsDNA molecule with a "bubble" in the middle. These bubble products run more slowly on the BA or a gel, causing them to appear as higher MW products. These samples could be run on a denaturing TBE-PAGE gel to see if the individual strands are indeed of the expected size.

The good news is that if the single strands are of the expected size you can still sequence these libraries with no problem, as they are denatured during cluster generation anyway. Quantification by QPCR will also be fine for the same reason.
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