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
Michael.
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
Michael.
Comment