# 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:\
![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-49149900b3cc4ac095106fbc8e4cd1c2075cd29b%2Fimage1.png?alt=media)

**Basespace:** the PrimaryAnalysisMetrics.csv file will not be present in the run files, but the metric will be displayed in the metrics tab:\
![](https://761066130-files.gitbook.io/~/files/v0/b/gitbook-x-prod.appspot.com/o/spaces%2FGM9W2DuBTgEXv1ClCm8H%2Fuploads%2Fgit-blob-e1465beb28ec36ee4fe69bc1de6470a97b731ff4%2Fimage2.png?alt=media\&token=9c08846a-7cc4-44d0-b388-84e5a1c6345b)

**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.

2. Although we do not recommend using %Occupancy for titrations, extreme % Occupancy, such as very high or very low, can indicate a loading concentration issue.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #3121), contact Illumina Technical Support* [*techsupport@illumina.com*](mailto:techsupport@illumina.com?subject=Question%2FFeedback%20Regarding%20Illumina%20Knowledge%20Article%20#000003121%20-%20Instrumentation%20\&body=Dear%20Illumina%20Technical%20Support,%0D%0A%0D%0A)*.* |