# 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 article.\
When sequencing libraries that have not been optimized on the NextSeq 1000/2000 with Standard SBS chemistry, **it is strongly recommended to perform titration runs**. *Even if a loading concentration is provided in the user guide, the loading concentration may need optimization.* The optimal loading concentration depends on the library type and insert size. Final loading concentrations for validated library types 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, 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. If the %Loading Concentration is <95%, increase the loading concentration in 100 pM increments for on board denature/dilute and 10 pM increments for manual denature/dilute over subsequent runs.

* For more information about finding % Loading Concentration, see the Knowledge Base Article: **Understanding Loading and Percent Loading Concentration on the NextSeq 1000/2000**

If the %Loading Concentration is less than 95%, increase loading concentration in 100 pM increments until 95% loading concentration is achieved.

\* 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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