# 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.
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.
\
\
\
| |
| :--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------: |
| *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)*.* |
---
# Agent Instructions: Querying This Documentation
If you need additional information that is not directly available in this page, you can query the documentation dynamically by asking a question.
Perform an HTTP GET request on the current page URL with the `ask` query parameter:
```
GET https://knowledge.illumina.com/instrumentation/nextseq-1000-2000/instrumentation-nextseq-1000-2000-troubleshooting-list/000003121.md?ask=
```
The question should be specific, self-contained, and written in natural language.
The response will contain a direct answer to the question and relevant excerpts and sources from the documentation.
Use this mechanism when the answer is not explicitly present in the current page, you need clarification or additional context, or you want to retrieve related documentation sections.