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

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

Hi,
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
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

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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
jdk787
josh kinman
 
Location: Austin

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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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