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

Background

Illumina recommends using the %Loading Concentration run metric alongside the %Occupied metric to optimize loading concentrations on the NextSeq 1000/2000. This is because the NextSeq 1000/2000 uses a unique patterned flow cell that requires a new metric to help accurately determine proper loading.

The %Loading Concentration was added to RTA3 release for NextSeq 1000/2000 Control Software v1.3 and above.

IMPORTANT NOTES

• This metric relies on the diversity and complexity of reads to estimate the appropriate loading concentration.

• Libraries containing low diversity or low complexity (ie, amplicon libraries) could potentially impact the accuracy of the %Loading concentration, so %Loading Concentration should not be used for libraries that have low diversity or high proportion of similar reads.

• For situations where %Loading Concentration cannot be used, it is best to review a combination of Percent Pass Filter (%PF), Q30, %Occupied, 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. The PrimaryAnalysisMetrics.csv file is only available once the run completes.

PrimaryAnalysisMetrics.csv from the on-instrument run folder: /usr/local/illumina/runs/[runfolder]

PrimaryAnalysisMetrics.csv file:

For runs in 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

For low diversity or low complexity libraries, use acombination of Percent Pass Filter (%PF), %Occupancy, Q30, and PhiX alignment to help with loading concentration optimization.

• For over-loaded runs, expect to see low passing filter, high %Occupancy, reduced quality scores and lower than expected PhiX alignment.

• For under-clustered runs, expect to see low passing filter, low %Occupancy, high quality and higher than expected PhiX alignment.

After reviewing metrics, increase or decrease loading concentration in 100 pM increments for titrations.

For any feedback or questions regarding this article (Illumina Knowledge Article #3121), contact Illumina Technical Support [e-mail].