Understanding loading and percent loading concentration on the NextSeq 1000/2000

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

1. 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.

1. 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 [e-mail].