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Old 06-08-2012, 09:08 AM   #1
Rosmano
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Default Bioanalyzer results - adapter dimers in ChIP-seq library samples

Hello all,

I've recently found this forum and wanted to ask for feedback about the quality of the attached ChIP-seq libraries.

The relevant information is:
Used Bioo Scientific NextFlex Barcode Adapters with a concentration of 0.6uM for a total amount of 10ng of DNA.
I size select the library to remove excess adapters and select a size range of templates around the 300bp size using a E-Gel Size select system
Following the size selection I did PCR amplification for 15 cycles of half of the sample.
Afterwards I did an Ampure bead purification to further clean up the sample of fragments with the wrong size.

However in most of my samples I still obtained adapter dimers peaks. Since adapter dimers are undesirable since they compete with the actual sample when sequencing in the Illumina Genome Analyzer that we are going to use I wanted to know if the amount of adapter dimers found in these samples is significant and I should redo the libraries maybe with a smaller amount of adapters.

I am also having issues with correctly determining the adequate amount of DNA in my samples: What I consistently observe is that input samples always have more DNA than IP samples in the E-Gel Recovery System even when I am always using 10ng of sample. So it's impossible to adjust the amount of DNA beforehand, if I am seeing variations only after the adapter ligation step.

So is any of those samples usable? I would like to know specifically if sample 2 can be used since I have a very limited amount of DNA of that material.

Thanks for the feedback.
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File Type: pdf ACF_8771-8773_DNA 1000_DE24802219_2012-06-08_10-22-09.pdf (455.1 KB, 253 views)
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Old 06-12-2012, 10:06 AM   #2
NextGenSeq
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Sample 2 is probably okay. We use the ultralow input kit from NuGEN and almost never see adapter dimers. How did you shear your DNA? The two peaks you get suggest over amplification but with 15 cycles you shouldn't see overamplification unless this is a very low complexity sample.
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Old 06-13-2012, 05:15 AM   #3
Rosmano
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So what's the size of the adapter-dimer peaks that make a sample bad for sequencing?
Is it when it is roughly the same size of the sample?
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Old 06-20-2012, 07:04 AM   #4
Rosmano
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Quote:
Originally Posted by NextGenSeq View Post
Sample 2 is probably okay. We use the ultralow input kit from NuGEN and almost never see adapter dimers. How did you shear your DNA? The two peaks you get suggest over amplification but with 15 cycles you shouldn't see overamplification unless this is a very low complexity sample.
I shear my DNA by sonication.
I repeated the PCR amplification (16 cycles this time) and the Ampure beads purification step with the other half of my samples. However, this time I changed the ratio of Ampure beads XL to DNA volume from 1,6:1 to 1,1:1 since I read here that decreasing the ratio can reduce the adapter dimers in the samples.

In fact I saw a reduction in the size of the adapter dimer peaks, however I still ahd a large amount of dimers. Are there any other steps that can be taken to reduce adapter dimers besides the steps I already mentioned?
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