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Old 04-29-2019, 10:16 PM   #1
Junior Member
Location: Melbourne, Australia

Join Date: Aug 2013
Posts: 8
Default Consistent low MiSeq cluster density and PhiX alignment with high quality reads (PF%)

Hi everyone,

I am struggling to understand an issue I am having with sequencing on an Illumina MiSeq. I have had success in the past with this exact protocol at a different institution - with another machine and reagents etc.

I am sequencing custom 20 pM ddRADseq libraries using a V3 600 cycle kit.
My workflow is;
library prep > pippin prep > AMPure > Tape station > Bioline library QC qPCR kit > dilute to 10 nM > side by side denature and dilute 10 nM library and PhiX to 20 pM > spike in 10% PhiX.

On my last run I got low cluster densities (365K/mm2) and low PhiX alignment (0.91% - aiming for 10%) with good sequence quality (91.51% >=Q30). This seems to be the way each run goes.

Any help would be appreciated

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