> For the complete documentation index, see [llms.txt](https://knowledge.illumina.com/llms.txt). Markdown versions of documentation pages are available by appending `.md` to page URLs; this page is available as [Markdown](https://knowledge.illumina.com/instrumentation/nextseq-1000-2000/instrumentation-nextseq-1000-2000-reference_material-list/000002435.md).

# NextSeq 1000/2000 loading optimization for Standard SBS kits

These recommendations are for **NextSeq 1000/2000 Standard SBS chemistry kits only**. When using NextSeq 1000/2000 XLEAP-SBS chemistry, reference the [NextSeq 1000/2000 Loading Optimization for XLEAP-SBS kits](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/NextSeq-1000-2000-XLEAP-Loading-Optimization/README.md) article. When sequencing third-party libraries or custom preparation protocol libraries with no recommended loading concentration, **it is strongly recommended to perform titration runs**. Note that even when a specific loading concentration is provided, it may still need further optimization since it depends on the library type and final fragment size. Final loading concentrations for validated Illumina libraries can be found in the [Illumina Denature and Dilute Guide](https://support-docs.illumina.com/IN/dnd-wizard/Content/dnd/landing.htm?ins=nextseq1k2k).\
**Starting Point for Standard SBS Loading concentrations**

For unlisted library types, start with a loading concentration of 650 pM for onboard denature/dilute (the same used for PhiX only runs), and 65 pM for manual denature/dilute, and optimize this concentration over subsequent runs to identify a loading concentration that consistently yields data that meets specifications.

To optimize loading concentration for high diversity libraries, use the %Loading Concentration metric in the PrimaryAnalysisMetrics.csv output file available after run completion. A review of the %Loading Concentration metric in conjunction with the %Occupancy and %aligned is recommended for optimal assessment.

If the %Loading Concentration is <95%, increase the loading concentration by 100 pM for onboard denature/dilute and 10 pM for manual denature/dilute over subsequent runs until it reaches 95%.

* For more information about finding % Loading Concentration, see the Knowledge Base Article: [Understanding loading and percent loading concentration on the NextSeq 1000/2000](https://github.com/illumina-swi/illumina-knowledge/tree/master/articles/Knowledge/Optimizing-cluster-density-on-the-NextSeq-2000/README.md)

For low base diversity libraries, the %Loading Concentration might not be accurate and using the %Occupancy with the %aligned alone might be a better option.

* If %Occupancy is <\~90% and %aligned is higher than the percentage of PhiX that was spiked in, run is underloaded.
* If %Occupancy is >\~95% and %aligned is lower than the percentage of PhiX that was spiked in, run is overloaded.

Titrations of 100 pM (for onboard denaturation protocol) or 10 pM (for manual denaturation protocol) are also recommended for low diversity libraries.

**Note:** When manually denaturing and diluting a library, the final loading concentration will be approximately 1/10th of that used for onboard denature/dilute.

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| *For any feedback or questions regarding this article (Illumina Knowledge Article #2435), 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)*.* |


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