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Old 02-21-2018, 11:45 AM   #1
dcheng1
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Location: blacksburg

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Default Tagmented DNA amplified very slowly

Hi,

I'm working on preparing ATAC-seq libraries. After tagmention, the DNA were amplified very slowly, and it takes 15+ cycles to reach exponential phase of pcr amplification.

I measured the amount of DNA by Nanodrop. Even if there are more than 1 ng of DNA, it still takes 17~20 cycles to get enough DNA for sequencing.

It seems like pcr were inhibited somehow. I've read that if Tn5 transposase were not removed from DNA fragments, it will inhibit pcr amplification. But I've tried proteinase K digestion, SDS treatment, adding EDTA, purifying by Zumo column. But none of them worked.

So here is my problem: I got more than 1 ng of tagmented DNA, but they were amplified very slowly. Any opinions are greatly appreciated!
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Old 02-21-2018, 01:28 PM   #2
nucacidhunter
Jafar Jabbari
 
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One cause could be low number of tagmented amplifiable fragments (fragmenst that have different tags in each end and are in a size range that PCR settings allows amplification). I wonder if you have looked at amplified fragments size profile.
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Old 02-21-2018, 01:54 PM   #3
dcheng1
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The amplified fragments are usually 300~1000 bp. Do you mean the tagmentation efficiency is too low?

I attached a tapestation image of amplified DNA.
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Old 02-21-2018, 03:26 PM   #4
nucacidhunter
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It is possible that in your cells accessible chromatin region frequency is low so less DNA is available for tagmentation.

Library profile does not look like ATAC-Seq as it lacks periodicity. Current library could be entirety from mitochondrial DNA. I would suggest reviewing your protocol.
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