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Old 11-13-2013, 05:05 AM   #1
chadinus
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Default Noisy and spiky Peaks

Dear All,

First I would like to thank you all for this wonderful forum. Very useful information indeed.

I am currently preparing Illumina libraries using Ethan's protocol (http://ethanomics.wordpress.com/chip...useq-adapters/).

The starting cells' number was around 400K. So I amplified the Genomic DNA obtained from ChIP experiment using Adli et al., 2011 protocol (http://www.nature.com/nprot/journal/....2011.402.html)

After the genomic DNA amplification procedure ends, I start with Ethan's protocol from the End repair step. I use 5 cycles for converting Y shaped adaptors. I excise the gel at 200-500 bp. Then I amplify the library using 11 cycles.

At the end I check the QC using agilent High sensitivity Chip (1:20 dilution). Some of the samples show good quality electropherogram while some others show noisy, pointy (spiky) and weird peaks. The range of the peak is correct but it has a weird appearance. I have ran all the samples that shows such peaks in a High sensitivity Bioanalyzer Chip (1:20) and have attached the PDF report of that run.

Have anyone seen such kind of peaks before after ChIP-seq library preparation? Can we sequence such libraries? What causes such appearance of peaks (as in the PDF report)?

I would really appreciate all your help and valuable suggestions...

With warmest regards,

Chadi
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File Type: pdf FHT131113(1).pdf (1.42 MB, 105 views)
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Old 11-16-2013, 04:33 PM   #2
Genohub
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In my experience you should be able to sequence these libraries without any problem. The spiky peaks are most likely due to your fragmentation method. How did you shear your samples?

- Genohub
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Old 11-18-2013, 05:58 PM   #3
chadinus
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Dear Genohub,

Thank you for your reply and help.

I agree with you. I also feel it is something related to fragmentation. I used probed sonicator for shearing the DNA. 35% pulse for 11 min. I feel the sonication should be enhanced.

Will try to sequence those libraries and will post the results in this thread as it might be useful for other readers later.

Thank you once again for your reply.

Chadi
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Old 03-23-2014, 08:23 AM   #4
vikas sharma
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Dear chadinus,

Did you sequenced these samples of yours, If so what was your result. I am interested to know.
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Old 03-26-2014, 10:17 PM   #5
chadinus
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Dear Vikas Sharma,

The libraries were of good quality.

However, I don't recommend Adli's et al. protocol for small scale ChIP as it will introduce additional primer sequences to the library. That will lead to lower mapping effeciency when you do the computational analysis. There are kits now that are available which can generate NGS libraries with a starting material of 50pg.

Regards,

Chadi
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Old 03-26-2014, 10:21 PM   #6
chadinus
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Dear Vikas Sharma,

The libraries were of good quality.

However, I don't recommend Adli's et al. protocol for small scale ChIP as it will introduce additional primer sequences to the library. That will lead to lower mapping effeciency when you do the computational analysis. There are kits now that are available which can generate NGS libraries with a starting material of 50pg.

Regards,

Chadi
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