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Old 08-11-2010, 07:07 AM   #1
krobison
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Default Better to risk low concentration library or further amplification?

Here's an issue which has arisen with some SureSelect processed libraries for the Illumina: after the hybridization the amount of library is barely within spec -- hence my question about quantitation.

So, the question is which path to take: push the libraries forward as is, with a risk of missing peak sequencer yield due to suboptimal cluster density, or run a few more (how many?) cycles of PCR to increase the concentration but also up the odds of getting PCR duplicates?
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Old 08-11-2010, 07:10 AM   #2
NextGenSeq
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It worked fine for me and I could barely detect it on my Nanodrop. We are in the process of evaluating the Kapa Q-PCR kit which I think the Broad Institute has switched to.
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Old 08-12-2010, 08:09 AM   #3
GW_OK
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Check out this thread.
You can prep a flowcell with a library that is <100pM with no problems.
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Old 08-12-2010, 02:40 PM   #4
lh3
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No, I think the expected PCR duplicate rate is irrelevant to the PCR cycles. The duplicate rate is determined by the number of molecules before amplification.
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