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Old 06-07-2011, 06:12 PM   #1
sehrrot
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Default Truseq library has dimer/trimer peak on bioanalyzer after amplfication

Dear all,

I've been making Illumina truseq DNA library and I've noticed dimer/trimer peak on bioanalyzer (high sensitivity assay) after PCR amplification. Is there anyone seeing this kind of result before?



To avoid this problem, I optimised PCR input, which was around 3ng total, and there was no dimer or trimer peak. Do you think it's gonna make less complex library? Is 3ng PCR input enough for final sequencing library?
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Last edited by sehrrot; 06-07-2011 at 06:16 PM.
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Old 06-07-2011, 07:40 PM   #2
ZAAB
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Hey:
Thanks for the reply on my separate post...
http://seqanswers.com/forums/showthr...3479#post43479

I'm just going to clone that 550nt beast and let illumina (and everyone here) know what it is.

I got around the issue by running 10 cycles of PCR, gel extracting the correct molecular weights, Quantifying via picogreen, and determined that 4 more PCR cycles would bring me up to the same amount of total DNA as I was seeing after the original 10 cycles--containing that beast 550 nt thing---
That extra 4 cycles brought my DNA amount up to similar amounts (no surprise there), but the PCR did not contain the 550nt band.

Off to cluster generation...
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Old 06-15-2011, 08:53 AM   #3
ashchin
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Hey,

How much amount of DNA are you starting with for librray prep?
We had the same issue which was resolved by reducing the amount of adapter ligated dna into pcr.
Also dilute the adapters accordingly. Try different ratios.

Thanks!
Ashwini
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Old 06-15-2011, 09:05 AM   #4
ZAAB
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I've taken this library prep through to completion with three different samples at this point, and I see that the ligation TIME is the screwy part...however I like the idea of decreasing adapter concentrations...
However, I am getting this nasty Laddering phenomenon, whereby I am getting concatenated DNA...again, Ligase concentration is the suspected culprit, even though the DNA should not be self-ligate-able, nor the adapter ends.
very strange.

Thanks for the suggestion to dilute adapter.
Take care.
Z
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Old 06-15-2011, 03:38 PM   #5
sehrrot
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Quote:
Originally Posted by ashchin View Post
Hey,

How much amount of DNA are you starting with for librray prep?
We had the same issue which was resolved by reducing the amount of adapter ligated dna into pcr.
Also dilute the adapters accordingly. Try different ratios.

Thanks!
Ashwini
Thanks Ashchin,

I started off fragmentation with 2.4 ug (calculated by QuBit BR) in 120 ul and then purified with AMPure beads. Then, remeasured the concentration with QuBit HS and got 50ng/ul as the Truseq protocol recommends.

I've tried different ligation time and also different PCR input several times. Different from the previous PE protocol, longer ligation time resulted in dimer issue. Also, too much PCR input did too. I strictly keep 10 minute ligation time and then put stop ligation buffer and also used 3-4 ng of input for amplficiatoin, which I am a bit worried about library complexity.

Anyway, it might be interesting to sequence dimer library and non-dimer library.
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