Understanding loading and percent loading concentration on the NextSeq 1000/2000
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Illumina recommends using the %Loading Concentration run metric to optimize loading concentration on the NextSeq 1000/2000. This is because the NextSeq 1000/2000 uses a unique patterned flow cell that required a new metric to help accurately determine proper loading.
%Loading Concentration was added to RTA3 release for NextSeq1000/2000 digital package v1.3 and greater
This method relies on the diversity of reads to estimate the proper the proper loading concentration. Libraries containing low diversity (ie, amplicon libraries) could potentially impact the accuracy of the %Loading concentration, so %Loading Concentration should not be used for low diversity libraries
For low diversity libraries, it is best to reference a combination of % Pass Filter, Q30, and PhiX alignment as seen below.
Identifying the %Loading Concentration for a run
The %Loading concentration for a sequencing run can be found within the PrimaryAnalysisMetrics.csv file in the run folder, as well as in run metrics in BaseSpace.
PrimaryAnalysisMetrics.csv* from the local run folder: /usr/local/Illumina/runs/[runfolder] *This file is only available once the run completes.
PrimaryAnalysisMetrics.csv file:
Basespace: the PrimaryAnalysisMetrics.csv file will not be present in the run files, but the metric will be displayed in the metrics tab:
Alternatives to %Loading Concentration
Use a combination of % Pass Filter, Q30, and PhiX alignment to help with loading concentration optimization.
If a run is overloaded: expect to see low passing filter, low quality and lower than expected PhiX alignment.
If a run is under loaded: expect to see low passing filter, high quality and higher than expected PhiX alignment.
Increase/decrease loading concentrations in 100 pM increments for titrations.
Although we do not recommend using %Occupancy for titrations, extreme % Occupancy, such as very high or very low, can indicate a loading concentration issue.
For any feedback or questions regarding this article (Illumina Knowledge Article #3121), contact Illumina Technical Support .
