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Old 07-30-2016, 01:15 AM   #1
Junior Member
Location: Uk

Join Date: May 2013
Posts: 2
Default low miseq clustering after kapa library quantification

Sorry if duplicating a thread.
I have performed 2 runs now with nebnext libraries (approx 350bp in size).
Both times I used kapa library quantity kits to quantify in triplicate over 2 dilutions (1000 & , 10000). Then I diluted multiplexed libs to total concentration of 4nm before following illumina protocol for library dilution to 15pm and loading on miseq.
On both runs the cluttering ws low (550 and 197 pf). I checked things like naoh concentration using ph paper and that was ok.
5% phi x was on both runs but miseq calculates final alignment of phi x as 20 and 25% respectively on each run suggesting my concentration of libraries was much lower than team 15pm I thought I loaded.
I had small amount of adaptor dimers in one library but surely this would not cause low clustering?
Could anyone give me suggestions on what I am doing wrong.
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Old 07-30-2016, 03:44 AM   #2
Jafar Jabbari
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238

Dimers cluster better than actual library. Higher ratio of PhiX indicates that it is either library itself or the quantification or both .
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Old 07-30-2016, 10:29 PM   #3
josh kinman
Location: Austin

Join Date: Apr 2014
Posts: 71

Since your PhiX read percentage is 4 to 5X higher than expected, I would suggest confirming your qPCR result with Qubit to make sure that something isn't wrong there.
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