Dear All,
I have recently observed very low cluster densities when sequencing libraries on the MiSeq.
Briefly, I had loaded the Flow cell with 17.2 pM libraries along with a 5% PhiX Spike in (Also 17.2pM). This I thought was very high concentration but went ahead since my last run gave me a low cluster density figures as well.
The Average size of my libraries are 350bp including the adaptor length. (so approx. 210-215bp insert size).
The quality of the data is great (98%>Q30; 94% PF). But, the output gives me 176K/mm2 cluster density with 5% PhiX as % aligned. This leads me to believe that my concentration calculations were accurate but the number of fragments sticking to the Flow Cell were way lesser than my previous experiences. The 2N NaOH stock was made fresh and accurately.
The only difference that I can think of from my previous runs and the current one (and penultimate run; where also I got low cluster densities) is that I used QIAEXII Gel Extraction instead of QiaQuick Gel Extraction.
Has anyone been in a similar situation? What could be the possible reasons for this? Does the gel extraction kit matter?
Thanks for your help.
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